Angiotensinogen (AGT) iRNA compositions and methods of use thereof

ABSTRACT

The present invention relates to RNAi agents, e.g., double stranded RNA (dsRNA) agents, targeting the AGT gene. The invention also relates to methods of using such RNAi agents to inhibit expression of an AGT gene and to methods of preventing and treating an AGT-associated disorder, e.g., high blood pressure.

RELATED APPLICATIONS

This application is a 35 § U.S.C. 111(a) continuation application whichclaims the benefit of priority to PCT/US2019/032150, filed on May 14,2019, which, in turn, claims the benefit of priority to U.S. ProvisionalApplication No. 62/671,094, filed on May 14, 2018, U.S. ProvisionalApplication No. 62/727,141, filed on Sep. 5, 2018, and U.S. ProvisionalApplication No. 62/816,996, filed on Mar. 12, 2019. The entire contentsof each of the foregoing applications are incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 29, 2020, isnamed 121301_08604_SL.txt and is 272,533 bytes in size.

BACKGROUND OF THE INVENTION

The renin-angiotensin-aldosterone system (RAAS) plays a crucial role inthe regulation of blood pressure. The RAAS cascade begins with therelease of angiotensinogen from the liver, and renin by thejuxtaglomerular cells of the kidney into the circulation. Reninsecretion is stimulated by several factors, including Na+ load in thedistal tubule, β-sympathetic stimulation, or reduced renal perfusion.Active renin in the plasma cleaves angiotensinogen (produced by theliver) to angiotensin I, which is then converted by circulating andlocally expressed angiotensin-converting enzyme (ACE) to angiotensin II.Most of the effects of angiotensin II on the RAAS are exerted by itsbinding to angiotensin II type 1 receptors (AT₁R), leading to arterialvasoconstriction, tubular and glomerular effects, such as enhanced Na+reabsorption or modulation of glomerular filtration rate. In addition,together with other stimuli such as adrenocorticotropin, anti-diuretichormone, catecholamines, endothelin, serotonin, and levels of Mg2+ andK+, AT₁R stimulation leads to aldosterone release which, in turn,promotes Na+ and K+ excretion in the renal distal convoluted tubule.

Dysregulation of the RAAS leading to, for example, excessive angiotensinII production or AT₁R stimulation results in hypertension which can leadto, e.g., increased oxidative stress, promotion of inflammation,hypertrophy, and fibrosis in the heart, kidneys, and arteries, andresult in, e.g., left ventricular fibrosis, arterial remodeling, andglomerulosclerosis.

Hypertension is the most prevalent, controllable disease in developedcountries, affecting 20-50% of adult populations. Hypertension is amajor risk factor for various diseases, disorders and conditions suchas, shortened life expectancy, chronic kidney disease, stroke,myocardial infarction, heart failure, aneurysms (e.g. aortic aneurysm),peripheral artery disease, heart damage (e.g., heart enlargement orhypertrophy) and other cardiovascular related diseases, disorders, orconditions. In addition, hypertension has been shown to be an importantrisk factor for cardiovascular morbidity and mortality accounting for,or contributing to, 62% of all strokes and 49% of all cases of heartdisease. In 2017, changes in the guidelines for diagnosis, prevention,and treatment of hypertension were developed providing goals for evenlower blood pressure to further decrease risk of development of diseasesand disorders associated with hypertension (see, e.g., Reboussin et al.

Systematic Review for the 2017ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for thePrevention, Detection, Evaluation, and Management of High Blood Pressurein Adults: A Report of the American College of Cardiology/American HeartAssociation Task Force on Clinical Practice Guidelines. J Am CollCardiol. 2017 Nov. 7. pii: S0735-1097(17)41517-8. doi:10.1016/j.jacc.2017.11.004; and Whelton et al. (2017ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for thePrevention, Detection, Evaluation, and Management of High Blood Pressurein Adults: A Report of the American College of Cardiology/American HeartAssociation Task Force on Clinical Practice Guidelines. J Am CollCardiol. 2017 Nov. 7. pii: S0735-1097(17)41519-1. doi:10.1016/j.jacc.2017.11.006).

Despite the number of anti-hypertensive drugs available for treatinghypertension, more than two-thirds of subjects are not controlled withone anti-hypertensive agent and require two or more anti-hypertensiveagents selected from different drug classes. This further reduces thenumber of subjects with controlled blood pressure as adherence isreduced and side-effects are increased with increasing numbers ofmedications.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which affect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a gene encoding angiotensinogen (AGT). The AGT may bewithin a cell, e.g., a cell within a subject, such as a human subject.

In an aspect, the invention provides a double stranded ribonucleic acid(dsRNA) agent for inhibiting expression of angiotensinogen (AGT),wherein the dsRNA agent comprises a sense strand and an antisense strandforming a double stranded region, wherein the sense strand comprises atleast 19 contiguous nucleotides from the nucleotide sequence of any oneof nucleotides 635-658, 636-658, 642-667, 642-664, 645-667, 1248-1273,1248-1272, 1248-1270, 1250-1272, 1251-1273, 1580-1602, 1584-1606,1587-1609, 1601-1623, 1881-1903, 2074-2097, 2074-2096, 2075-2097,2080-2102, 2272-2294, 2276-2298, 2281-2304, 2281-2303, or 2282-2304 ofSEQ ID NO:1 and the antisense strand comprises at least 19 contiguousnucleotides from the nucleotide sequence of SEQ ID NO:2.

In certain embodiments, the sense strand comprises at least 21contiguous nucleotides of any one of nucleotides 635-658, 636-658,642-667, 642-664, 645-667, 1248-1273, 1248-1272, 1248-1270, 1250-1272,1251-1273, 1580-1602, 1584-1606, 1587-1609, 1601-1623, 1881-1903,2074-2097, 2074-2096, 2075-2097, 2080-2102, 2272-2294, 2276-2298,2281-2304, 2281-2303, or 2282-2304 of SEQ ID NO:1. In certainembodiments, the antisense strand comprises at least 21 contiguousnucleotides from the nucleotide sequence of SEQ ID NO:2.

In certain embodiments, the antisense strand comprises at least 19contiguous nucleotides from any one of the antisense strand nucleotidesequences of a duplex selected from the group consisting of AD-85481,AD-84701, AD-84703, AD-84704, AD-84705, AD-84707, AD-84715, AD-84716,AD-84739, AD-84741, AD-84746, AD-85432, AD-85434, AD-85435, AD-85436,AD-85437, AD-85438, AD-85441, AD-85442, AD-85443, AD-85444, AD-85446,AD-85447, AD-85482, AD-85485, AD-85493, AD-85496, AD-85504, AD-85517,AD-85519, AD-85524, AD-85622, AD-85623, AD-85625, AD-85626, AD-85634,AD-85635, AD-85637, AD-85655, AD-126306, AD-126307, AD-126308,AD-126310, AD133360, AD-133361, AD-133362, AD-133374, and AD-133385. Incertain embodiments, the sense strand comprises at least 19 contiguousnucleotides from any one of the sense strand nucleotide sequences of aduplex selected from the group consisting of AD-85481, AD-84701,AD-84703, AD-84704, AD-84705, AD-84707, AD-84715, AD-84716, AD-84739,AD-84741, AD-84746, AD-85432, AD-85434, AD-85435, AD-85436, AD-85437,AD-85438, AD-85441, AD-85442, AD-85443, AD-85444, AD-85446, AD-85447,AD-85482, AD-85485, AD-85493, AD-85496, AD-85504, AD-85517, AD-85519,AD-85524, AD-85622, AD-85623, AD-85625, AD-85626, AD-85634, AD-85635,AD-85637, AD-85655, AD-126306, AD-126307, AD-126308, AD-126310,AD133360, AD-133361, AD-133362, AD-133374, and AD-133385. In certainembodiments, the sense and antisense strands comprise nucleotidesequences of a duplex selected from the group consisting of AD-85481,AD-84701, AD-84703, AD-84704, AD-84705, AD-84707, AD-84715, AD-84716,AD-84739, AD-84741, AD-84746, AD-85432, AD-85434, AD-85435, AD-85436,AD-85437, AD-85438, AD-85441, AD-85442, AD-85443, AD-85444, AD-85446,AD-85447, AD-85482, AD-85485, AD-85493, AD-85496, AD-85504, AD-85517,AD-85519, AD-85524, AD-85622, AD-85623, AD-85625, AD-85626, AD-85634,AD-85635, AD-85637, AD-85655, AD-126306, AD-126307, AD-126308,AD-126310, AD133360, AD-133361, AD-133362, AD-133374, and AD-133385.

In certain embodiments, the antisense strand comprises at least 19contiguous nucleotides from nucleotide sequence of the antisense strandof AD-85481 (5′-UGUACUCUCAUUGUGGAUGACGA-3′ (SEQ ID NO: 9)). In certainembodiments, the sense strand comprises at least 19 contiguousnucleotides from the nucleotide sequences of the sense strand ofAD-85481 (5′-GUCAUCCACAAUGAGAGUACA-3′ (SEQ ID NO: 10)). In certainembodiments, the sense and antisense strands comprise the nucleotidesequences of the sense and antisense strands of AD-85481(5′-UGUACUCUCAUUGUGGAUGACGA-3′ (SEQ ID NO: 9) and5′-GUCAUCCACAAUGAGAGUACA-3′ (SEQ ID NO: 10)).

In certain embodiments, the dsRNA agent comprises at least one modifiednucleotide. In certain embodiments, substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand comprise a modification. In certain embodiments, all ofthe nucleotides of the sense strand and all of the nucleotides of theantisense strand comprise a modification. In certain embodiments, atleast one of the modified nucleotides is selected from the group of adeoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an unlockednucleotide, a conformationally restricted nucleotide, a constrainedethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide,a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide,2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide,a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a5′-phosphate mimic, a thermally destabilizing nucleotide, a glycolmodified nucleotide (GNA), and a 2-O—(N-methylacetamide) modifiednucleotide; and combinations thereof. In certain embodiments, themodifications on the nucleotides are selected from the group consistingof LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl,2′-fluoro, 2′-deoxy, 2′-hydroxyl, GNA, and combinations thereof. Incertain embodiments, the modifications on the nucleotides are2′-O-methyl or 2′-fluoro modifications. In certain embodiments, at leastone of the modified nucleotides is selected from the group consisting ofa deoxy-nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a glycol modifiednucleotide (GNA), and a 2-O—(N-methylacetamide) modified nucleotide; andcombinations thereof. In certain embodiments, at least one of thenucleotide modification is a thermally destabilizing nucleotidemodification. In certain embodiments, the thermally destabilizingnucleotide modification is selected from the group consisting of of anabasic modification; a mismatch with the opposing nucleotide in theduplex; and destabilizing sugar modification, a 2′-deoxy modification,an acyclic nucleotide, an unlocked nucleic acids (UNA), and a glycerolnucleic acid (GNA) In certain embodiments, the double stranded region is19-21 nucleotides in length. In certain embodiments, the double strandedregion is 21 nucleotides in length. In certain embodiments, each strandof the dsRNA agent is independently no more than 30 nucleotides inlength. In certain embodiments, at least one strand of the dsRNA agentcomprises a 3′ overhang of at least 1 nucleotide or at least 2nucleotides.

In certain embodiments, dsRNA agent further comprises a ligand. Incertain embodiments, the ligand is conjugated to the 3′ end of the sensestrand of the dsRNA agent. In certain embodiments, the ligand is anN-acetylgalactosamine (GaNAc) derivative, e.g., wherein the ligand is

In certain embodiments, the dsRNA agent is conjugated to the ligand asshown in the following schematic

and, wherein X is O or S, e.g., wherein the X is O.

In certain embodiments, the invention provides a dsRNA agent, whereinthe antisense strand comprises a region of complementarity to an mRNAencoding human AGT, wherein the region of complementarity comprises atleast 19 nucleotides one of the antisense strand sequences of a duplexselected from the group consisting of AD-85481, AD-84701, AD-84703,AD-84704, AD-84705, AD-84707, AD-84715, AD-84716, AD-84739, AD-84741,AD-84746, AD-85432, AD-85434, AD-85435, AD-85436, AD-85437, AD-85438,AD-85441, AD-85442, AD-85443, AD-85444, AD-85446, AD-85447, AD-85482,AD-85485, AD-85493, AD-85496, AD-85504, AD-85517, AD-85519, AD-85524,AD-85622, AD-85623, AD-85625, AD-85626, AD-85634, AD-85635, AD-85637,AD-85655, AD-126306, AD-126307, AD-126308, AD-126310, AD133360,AD-133361, AD-133362, AD-133374, and AD-133385. In certain embodiments,the antisense strand comprises a region of complementarity to an mRNAencoding human AGT, wherein the region of complementarity comprises anyone of the antisense strand sequences of a duplex selected from thegroup consisting of AD-85481, AD-84701, AD-84703, AD-84704, AD-84705,AD-84707, AD-84715, AD-84716, AD-84739, AD-84741, AD-84746, AD-85432,AD-85434, AD-85435, AD-85436, AD-85437, AD-85438, AD-85441, AD-85442,AD-85443, AD-85444, AD-85446, AD-85447, AD-85482, AD-85485, AD-85493,AD-85496, AD-85504, AD-85517, AD-85519, AD-85524, AD-85622, AD-85623,AD-85625, AD-85626, AD-85634, AD-85635, AD-85637, AD-85655, AD-126306,AD-126307, AD-126308, AD-126310, AD133360, AD-133361, AD-133362,AD-133374, and AD-133385. In certain embodiments, the region ofcomplementarity consists of any one of the antisense strand sequences ofa duplex selected from the group consisting of AD-85481, AD-84701,AD-84703, AD-84704, AD-84705, AD-84707, AD-84715, AD-84716, AD-84739,AD-84741, AD-84746, AD-85432, AD-85434, AD-85435, AD-85436, AD-85437,AD-85438, AD-85441, AD-85442, AD-85443, AD-85444, AD-85446, AD-85447,AD-85482, AD-85485, AD-85493, AD-85496, AD-85504, AD-85517, AD-85519,AD-85524, AD-85622, AD-85623, AD-85625, AD-85626, AD-85634, AD-85635,AD-85637, AD-85655, AD-126306, AD-126307, AD-126308, AD-126310,AD133360, AD-133361, AD-133362, AD-133374, and AD-133385.

In certain embodiments, the invention provides a dsRNA agent, whereinthe antisense strand comprises the chemically modified nucleotidesequence of a duplex selected from the group consisting of AD-85481,AD-84701, AD-84703, AD-84704, AD-84705, AD-84707, AD-84715, AD-84716,AD-84739, AD-84741, AD-84746, AD-85432, AD-85434, AD-85435, AD-85436,AD-85437, AD-85438, AD-85441, AD-85442, AD-85443, AD-85444, AD-85446,AD-85447, AD-85482, AD-85485, AD-85493, AD-85496, AD-85504, AD-85517,AD-85519, AD-85524, AD-85622, AD-85623, AD-85625, AD-85626, AD-85634,AD-85635, AD-85637, AD-85655, AD-126306, AD-126307, AD-126308,AD-126310, AD133360, AD-133361, AD-133362, AD-133374, and AD-133385.

In certain embodiments, the invention provides a dsRNA agent, whereinthe antisense strand comprises the chemically modified nucleotidesequence of the duplex AD-85481(5′-usGfsuac(Tgn)cucauugUfgGfaugacsgsa-3′ (SEQ ID NO: 11)) wherein a, c,g, and u are 2′-O-methyladenosine-3′-phosphate,2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and2′-O-methyluridine-3′-phosphate, respectively; Af, Cf, Gf, and Uf are2′-O-fluoroadenosine-3′-phosphate, 2′-O-fluorocytidine-3′-phosphate,2′-O-fluoroguanosine-3′-phosphate, and 2′-O-fluorouridine-3′-phosphate,respectively; dT is a deoxy-thymine; s is a phosphorothioate linkage;and (Tgn) is thymidine-glycol nucleic acid (GNA) S-isomer.

In certain embodiments, the invention provides a dsRNA agent, whereinthe antisense strand and the sense strand comprise the chemicallymodified nucleotide sequences of a duplex selected from the groupconsisting of AD-85481, AD-84701, AD-84703, AD-84704, AD-84705,AD-84707, AD-84715, AD-84716, AD-84739, AD-84741, AD-84746, AD-85432,AD-85434, AD-85435, AD-85436, AD-85437, AD-85438, AD-85441, AD-85442,AD-85443, AD-85444, AD-85446, AD-85447, AD-85482, AD-85485, AD-85493,AD-85496, AD-85504, AD-85517, AD-85519, AD-85524, AD-85622, AD-85623,AD-85625, AD-85626, AD-85634, AD-85635, AD-85637, AD-85655, AD-126306,AD-126307, AD-126308, AD-126310, AD133360, AD-133361, AD-133362,AD-133374, and AD-133385.

In certain embodiments, the invention provides a dsRNA agent, whereinthe antisense strand and the sense strand comprise the chemicallymodified nucleotide sequences of the duplex AD-85481(5′-usGfsuac(Tgn)cucauugUfgGfaugacsgsa-3′ (SEQ ID NO: 11) and5′-gsuscaucCfaCfAfAfugagaguaca-3′ (SEQ ID NO: 12)) wherein a, c, g, andu are 2′-O-methyladenosine-3′-phosphate,2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and2′-O-methyluridine-3′-phosphate, respectively; Af, Cf, Gf, and Uf are2′-O-fluoroadenosine-3′-phosphate, 2′-O-fluorocytidine-3′-phosphate,2′-O-fluoroguanosine-3′-phosphate, and 2′-O-fluorouridine-3′-phosphate,respectively; dT is a deoxy-thymine; s is a phosphorothioate linkage;and (Tgn) is thymidine-glycol nucleic acid (GNA) S-isomer; and whereinthe 3′-end of the sense strand is optionally conjugated to anN-[tris(GaNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (L96) ligand.

In certain embodiments, the invention provides a dsRNA agent, whereinthe antisense strand and the sense strand consist of the chemicallymodified nucleotide sequences of the duplex AD-85481(5′-usGfsuac(Tgn)cucauugUfgGfaugacsgsa-3′ (SEQ ID NO: 11) and5′-gsuscaucCfaCfAfAfugagaguaca-3′ (SEQ ID NO: 12)), wherein the 3′-endof the sense strand is conjugated to anN-[tris(GaNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (L96) ligand,wherein a, c, g, and u are 2′-O-methyladenosine-3′-phosphate,2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and2′-O-methyluridine-3′-phosphate, respectively; Af, Cf, Gf, and Uf are2′-O-fluoroadenosine-3′-phosphate, 2′-O-fluorocytidine-3′-phosphate,2′-O-fluoroguanosine-3′-phosphate, and 2′-O-fluorouridine-3′-phosphate,respectively; dT is a deoxy-thymine; s is a phosphorothioate linkage;and (Tgn) is thymidine-glycol nucleic acid (GNA) S-isomer.

In certain embodiments, the double stranded region of the dsRNA agent isabout 19-30 nucleotide pairs in length, about 19-25 nucleotide pairs inlength, about 23-27 nucleotide pairs in length, about 19-23 nucleotidepairs in length, about 21-23 nucleotide pairs in length.

In certain embodiments, each strand of the dsRNA agent is independently19-30 nucleotides in length.

In certain embodiments, the ligand is one or more GaNAc derivativesattached through a monovalent, bivalent, or trivalent branched linker.

In certain embodiments, the dsRNA agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage. Incertain embodiments, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 3′-terminus of one strand. In certainembodiments, the strand is the antisense strand. In certain embodiments,the strand is the sense strand.

In certain embodiments, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand. In certainembodiments, the strand is the antisense strand. In certain embodiments,the strand is the sense strand. In certain embodiments, thephosphorothioate or methylphosphonate internucleotide linkage is at theboth the 5′- and 3′-terminus of one strand. In certain embodiments, thestrand is the antisense strand.

In certain embodiments, the dsRNA agent at the 1 position of the 5′-endof the antisense strand of the duplex comprises a base pair that is anAU base pair.

In certain embodiments, the dsRNA agent comprises a sense strand has atotal of 21 nucleotides and an antisense strand has a total of 23nucleotides.

In an aspect, the invention provides a cell containing the dsRNA agentof the invention.

In an aspect, the invention provides a pharmaceutical composition forinhibiting expression of a gene encoding AGT comprising the dsRNA agentof the invention. In certain embodiments, the pharmaceutical compositioncomprises the dsRNA agent and a lipid formulation.

In an aspect, the invention provides a method of inhibiting expressionof an AGTgene in a cell, the method comprising:

(a) contacting the cell with the dsRNA agent or a pharmaceuticalcomposition of the invention; and

(b) maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of the AGT gene, therebyinhibiting expression of the AGT gene in the cell.

In certain embodiments, the cell is within a subject. In certainembodiments, the subject is a human. In certain embodiments, the subjecthas been diagnosed with an AGT-associated disorder.

In certain embodiments, the AGT-associated disorder is selected fromhigh blood pressure, hypertension, borderline hypertension, primaryhypertension, secondary hypertension isolated systolic or diastolichypertension, pregnancy-associated hypertension, diabetic hypertension,resistant hypertension, refractory hypertension, paroxysmalhypertension, renovascular hypertension, Goldblatt hypertension, ocularhypertension, glaucoma, pulmonary hypertension, portal hypertension,systemic venous hypertension, systolic hypertension, labilehypertension; hypertensive heart disease, hypertensive nephropathy,atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy,diabetic retinopathy, chronic heart failure, cardiomyopathy, diabeticcardiac myopathy, glomerulosclerosis, coarctation of the aorta, aorticaneurism, ventricular fibrosis, heart failure, myocardial infarction,angina, stroke, renal disease, renal failure, systemic sclerosis,intrauterine growth restriction (IUGR), fetal growth restriction,obesity, liver steatosis/fatty liver, non-alcoholic Steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance,type 2 diabetes (non-insulin dependent diabetes), and metabolicsyndrome.

In certain embodiments, the subject has a systolic blood pressure of atleast 130 mm Hg or a diastolic blood pressure of at least 80 mm Hg. Incertain embodiments, the subject has a systolic blood pressure of atleast 140 mm Hg and a diastolic blood pressure of at least 80 mm Hg. Incertain embodiments, the subject is part of a group susceptible to saltsensitivity, is overweight, is obese, or is pregnant.

In certain embodiments, contacting the cell with the dsRNA agentinhibits the expression of AGT by at least 50%, 60%, 70%, 80%, 90%, 95%(e.g., as compared to the level of expression of AGT prior to firstcontacting the cell with the dsRNA agent; e.g., prior to administrationof a first dose of the dsRNA agent to the subject). In certainembodiments, inhibiting expression of AGT decreases an AGT protein levelin a subject serum sample(s) by at least 50%, 60%, 70%, 80%, 90%, or95%, e.g., as compared to the level of expression of AGT prior to firstcontacting the cell with the dsRNA agent.

In an aspect, the invention provides a method of treating a anAGT-associated disorder in a subject, comprising administering to thesubject the dsRNA agent or the pharmaceutical composition of theinvention, thereby treating the AGT-associated disorder in the subject.In certain embodiments, the subject has a systolic blood pressure of atleast 130 mm Hg or a diastolic blood pressure of at least 80 mm Hg. Incertain embodiments, the subject has a systolic blood pressure of atleast 140 mm Hg and diastolic blood pressure of at least 80 mm Hg. Incertain embodiments, the subject is human. In certain embodiments,subject is part of a group susceptible to salt sensitivity, isoverweight, is obese, or is pregnant.

In certain embodiments of the invention, the dsRNA agent is administeredat a dose of about 0.01 mg/kg to about 50 mg/kg. In certain embodiments,the dsRNA agent is administered to the subject subcutaneously. Incertain embodiments, the level of AGT is measured in the subject. Incertain embodiments, the level of AGT in the subject is an AGT proteinlevel in a subject blood sample(s), serum sample(s), or urine sample(s).

In certain embodiments, an additional therapeutic agent for treatment ofhypertension is administered to the subject. In certain embodiments, theadditional therapeutic agent is selected from the group consisting of ofa diuretic, an angiotensin converting enzyme (ACE) inhibitor, anangiotensin II receptor antagonist, a beta-blocker, a vasodialator, acalcium channel blocker, an aldosterone antagonist, an alpha2-agonist, arenin inhibitor, an alpha-blocker, a peripheral acting adrenergic agent,a selective D1 receptor partial agonist, a nonselective alpha-adrenergicantagonist, a synthetic, and steroidal antimineralocorticoid agent; or acombination of any of the foregoing, and a hypertension therapeuticagent formulated as a combination of agents. In certain embodiments, theadditional therapeutic agent comprises an angiotensin II receptorantagonist, e.g., losartan, valsartan, olmesartan, eprosartan, andazilsartan. In certain embodiments, the additional therapeutic agent isan angiotensin receptor-neprilysin inhibitor (ARNi), e.g., Entresto®,sacubitril/valsartan; or an endothelin receptor antagonist (ERA), e.g.,sitaxentan, ambrisentan, atrasentan, BQ-123, zibotentan, bosentan,macitentan, and tezosentan.

The invention also provides uses of the dsRNA agents and thepharmaceutical compositions provided herein for treatment of anAGT-associated disorder. In certain embodiments, the uses include any ofthe methods provided by the invention.

The invention provides kits comprising a dsRNA agent of the invention.In certain embodiments, the invention provides kits for practicing amethod of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing serum AGT protein levels in cynomolgusmonkeys (n=3 per group) treated with a single 3 mg/kg dose of theindicated siRNAs. AGT levels are shown as a percent of AGT level priorto treatment.

FIG. 1B is a graph showing serum AGT protein levels in cynomolgusmonkeys (n=3 per group) treated with a single 0.3 mg/kg, 1 mg/kg, or 3mg/kg dose of AD-85481 or AD-67327 on day 1. AGT levels are shown as apercent of AGT level prior to treatment.

FIG. 1C is a graph showing serum AGT protein levels in cynomolgusmonkeys (n=3 per group) administered a 1 mg/kg dose of AD-85481 orAD-67327 once every four weeks for three doses. AGT levels are shown asa percent of AGT level prior to treatment.

FIGS. 2A-2G show the results of a various parameters in a study ofspontaneously hypertensive rats (n=9 per group) treated with a vehicle,valsartan (31 mg/kg/day), a rat specific AGT-siRNA (10 mg/kg q2w),captopril (100 mg/kg/day), valsartan and captopril, or valsartan andAGT-siRNA.

FIG. 2A shows plasma AGT levels at the start (solid bars) and end(stippled bars) of the study (at four weeks).

FIG. 2B shows daily blood pressure readings compared to baseline.

FIG. 2C is a graph showing heart weight: tibial length ratios.

FIG. 2D is a graph showing plasma renin activity level at the start(solid bars) and end (stippled bars) of the study (at four weeks).

FIG. 2E is a graph of heart weight:tibial length graphed against meanarterial pressure (MAP) in mm Hg.

FIG. 2F is a graph of cardiomyocyte size.

FIG. 2G is a graph of N-terminal pro b-type natriuretic peptide(NT-proBNP) levels.

FIG. 3 is a graph showing urine AGT levels in the spontaneouslyhypertensive rat study.

FIG. 4A is a graph showing the level of blood Ang I in the spontaneoushypertensive rat study.

FIG. 4B is a graph showing the level of blood Ang II in the spontaneoushypertensive rat study.

FIG. 4C is a graph showing the ratio of blood Ang II to blood Ang I inthe spontaneous hypertensive rat study.

FIG. 5A is a graph showing the level of renal Ang I in the spontaneoushypertensive rat study.

FIG. 5B is a graph showing the level of renal Ang II in the spontaneoushypertensive rat study.

FIG. 5C is a graph showing the ratio of renal Ang II to renal Ang I inthe spontaneous hypertensive rat study.

FIG. 6A is a graph showing the level of angiotensin receptor 1a in thekidney cortex and medulla in the spontaneous rat hypertensive study.

FIG. 6B is a graph showing the level of angiotensin 1b receptor in thekidney cortex and medulla in the spontaneous rat hypertensive study.

FIG. 6C is a graph showing the level of ACE in the kidney cortex andmedulla in the spontaneous rat hypertensive study.

FIG. 7 is a graph showing urinary volume at baseline and at 4 weeksafter the start of treatment in the spontaneous rat hypertensive study.

FIG. 8A is a graph showing average body weights of high fat fed dietinduced obesity (DIO) mice or normal chow fed mice (n=5 per group)treated with either an AGT dsRNA agent or PBS.

FIG. 8B is a graph showing terminal liver, adipose, and muscle weights(n=5 per group) of high fat fed diet induced obesity (DIO) mice ornormal chow fed mice treated with either an AGT dsRNA agent or PBS.

FIG. 9A is a graph showing the changes in plasma glucose levels (mg/dL)in high fat fed diet induced obesity (DIO) mice or normal chow fed mice(n=5 per group) treated with either an AGT dsRNA agent or PBS at week 0prior to first treatment dose.

FIG. 9B is a graph showing the plasma glucose levels (mg/dL) in high fatfed diet induced obesity (DIO) mice or normal chow fed mice (n=5 pergroup) treated with either an AGT dsRNA agent or PBS at week 6 of theexperiment.

FIG. 9C is a graph showing the plasma glucose levels (mg/dL) in high fatfed diet induced obesity (DIO) mice or normal chow fed mice (n=5 pergroup) treated with either an AGT dsRNA agent or PBS at week 12 of theexperiment.

FIG. 10A is a graph showing average body weights of high fat highfructose (HF HFr) fed mice treated with either an AGT dsRNA agent orPBS, or normal chow fed (LFD) mice.

FIG. 10B is a graph showing average cumulative weight gain of high fathigh fructose (HF HFr) fed mice treated with either an AGT dsRNA agentor PBS, or normal chow fed (LFD) mice.

FIGS. 11A-11C are graphs showing serum liver enzymes in high fat highfructose (HF HFr) fed mice treated with either an AGT dsRNA agent orPBS, or normal chow fed (LFD) mice at week 20 of the experiment.

FIG. 11A is a graph showing alanine transaminase (ALT) levels in highfat high fructose (HF HFr) fed mice treated with either an AGT dsRNAagent or PBS, or normal chow fed (LFD) mice.

FIG. 11B is a graph showing aspartate transaminase (AST) levels in highfat high fructose (HF HFr) fed mice treated with either an AGT dsRNAagent or PBS, or normal chow fed (LFD) mice.

FIG. 11C is a graph showing glutamate dehydrogenase (GLDH) levels inhigh fat high fructose (HF HFr) fed mice treated with either an AGTdsRNA agent or PBS, or normal chow fed (LFD) mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an AGT gene. The gene may be within a cell, e.g., a cellwithin a subject, such as a human. The use of these iRNAs enables thetargeted degradation of mRNAs of the corresponding gene (AGT gene) inmammals.

The iRNAs of the invention have been designed to target the human AGTgene, including portions of the gene that are conserved in the AGTorthologs of other mammalian species. Without intending to be limited bytheory, it is believed that a combination or sub-combination of theforegoing properties and the specific target sites or the specificmodifications in these iRNAs confer to the iRNAs of the inventionimproved efficacy, stability, potency, durability, and safety.

Accordingly, the present invention provides methods for treating andpreventing an AGT-associated disorder, e.g., hypertension, using iRNAcompositions which effect the RNA-induced silencing complex(RISC)-mediated cleavage of RNA transcripts of an AGT gene.

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is up to about 30 nucleotides or less in length,e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22,19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23,20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or21-22 nucleotides in length, which region is substantially complementaryto at least part of an mRNA transcript of an AGT gene.

In certain embodiments, one or both of the strands of the doublestranded RNAi agents of the invention is up to 66 nucleotides in length,e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length,with a region of at least 19 contiguous nucleotides that issubstantially complementary to at least a part of an mRNA transcript ofan AGT gene. In some embodiments, such iRNA agents having longer lengthantisense strands preferably may include a second RNA strand (the sensestrand) of 20-60 nucleotides in length wherein the sense and antisensestrands form a duplex of 18-30 contiguous nucleotides.

The use of iRNAs of the invention enables the targeted degradation ofmRNAs of the corresponding gene (AGT gene) in mammals. Using in vitroand in vivo assays, the present inventors have demonstrated that iRNAstargeting an AGT gene can mediate RNAi, resulting in significantinhibition of expression of AGT. Inhibition of expression of AGT in sucha subject will prevent or treat development of a AGT-associateddisorder, e.g., hypertension. Thus, methods and compositions includingthese iRNAs are useful for preventing and treating a subject susceptibleto or diagnosed with an AGT-associated disorder, e.g., hypertension. Themethods and compositions herein are useful for reducing the level of AGTin a subject.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of an AGT geneas well as compositions, uses, and methods for treating subjects thatwould benefit from reduction of the expression of an AGT gene, e.g.,subjects susceptible to or diagnosed with an AGT-associated disorder,e.g., hypertension.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise. Forexample, “sense strand or antisense strand” is understood as “sensestrand or antisense strand or sense strand and antisense strand.”

The term “about” is used herein to mean within the typical ranges oftolerances in the art. For example, “about” can be understood as about 2standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means ±5%. When about is presentbefore a series of numbers or a range, it is understood that “about” canmodify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understoodto include the number adjacent to the term “at least”, and allsubsequent numbers or integers that could logically be included, asclear from context. For example, the number of nucleotides in a nucleicacid molecule must be an integer. For example, “at least 19 nucleotidesof a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21nucleotides have the indicated property. When at least is present beforea series of numbers or a range, it is understood that “at least” canmodify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the valueadjacent to the phrase and logical lower values or integers, as logicalfrom context, to zero. For example, a duplex with an overhang of “nomore than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “nomore than” is present before a series of numbers or a range, it isunderstood that “no more than” can modify each of the numbers in theseries or range. As used herein, ranges include both the upper and lowerlimit.

In the event of a conflict between a sequence and its indicated site ona transcript or other sequence, the nucleotide sequence recited in thespecification takes precedence.

As used herein, “angiotensinogen,” used interchangeably with the term“AGT” refers to the well-known gene and polypeptide, also known in theart as Serpin Peptidase Inhibitor, Clade A, Member 8; Alpha-1Antiproteinase; Antitrypsin; SERPINA8; Angiotensin I; Serpin A8;Angiotensin II; Alpha-1 Antiproteinase angiotensinogen; antitrypsin;pre-angiotensinogen2; ANHU; Serine Proteinase Inhibitor; and CysteineProteinase Inhibitor.

The term “AGT” includes human AGT, the amino acid and complete codingsequence of which may be found in for example, GenBank Accession No.GI:188595658 (NM_000029.3; SEQ ID NO:1); Macacafascicularis AGT, theamino acid and complete coding sequence of which may be found in forexample, GenBank Accession No. GI: 90075391 (AB170313.1: SEQ ID NO:3);mouse (Mus musculus) AGT, the amino acid and complete coding sequence ofwhich may be found in for example, GenBank Accession No. GI: 113461997(NM_007428.3; SEQ ID NO:5); and rat AGT (Rattus norvegicus) AGT theamino acid and complete coding sequence of which may be found in forexample, for example GenBank Accession No. GI:51036672 (NM_134432; SEQID NO:7).

Additional examples of AGT mRNA sequences are readily available usingpublicly available databases, e.g., GenBank, UniProt, OMIM, and theMacaca genome project web site.

The term “AGT,” as used herein, also refers to naturally occurring DNAsequence variations of the AGT gene, such as a single nucleotidepolymorphism (SNP) in the AGT gene. Exemplary SNPs may be found in thedbSNP database available atwww.ncbi.nlm.nih.gov/projects/SNP/snp-_refcgi?geneId=183. Non-limitingexamples of sequence variations within the AGT gene include, forexample, those described in U.S. Pat. No. 5,589,584, the entire contentsof which are incorporated herein by reference. For example, sequencevariations within the AGT gene may include as a C→T at position −532(relative to the transcription start site); a G→A at position −386; aG→A at position −218; a C→T at position −18; a G→A and a A→C at position−6 and −10; a C→T at position +10 (untanslated); a C→T at position +521(T174M); a T-C at position +597 (P199P); a T-C at position +704 (M235T;also see, e.g., Reference SNP (refSNP) Cluster Report: rs699, availableat www.ncbi.nlm.nih.gov/SNP); a A-G at position +743 (Y248C); a C→T atposition +813 (N271N); a G→A at position +1017 (L339L); a C-A atposition +1075 (L359M); and/or a G→A at position +1162 (V388M).

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an AGT gene, including mRNA that is a product of RNA processing of aprimary transcription product. The target portion of the sequence willbe at least long enough to serve as a substrate for iRNA-directedcleavage at or near that portion of the nucleotide sequence of an mRNAmolecule formed during the transcription of an AGT gene. In oneembodiment, the target sequence is within the protein coding region ofAGT.

The target sequence may be from about 19-36 nucleotides in length, e.g.,preferably about 19-30 nucleotides in length. For example, the targetsequence can be about 19-30 nucleotides, 19-30, 19-29, 19-28, 19-27,19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28,20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28,21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine, and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 2). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of an AGT gene in a cell, e.g., a cell within a subject,such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., an AGTtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., an AGT gene. Accordingly, the term“siRNA” is also used herein to refer to an iRNA as described above.

In certain embodiments, the RNAi agent may be a single-stranded siRNA(ssRNAi) that is introduced into a cell or organism to inhibit a targetmRNA. Single-stranded RNAi agents bind to the RISC endonuclease,Argonaute 2, which then cleaves the target mRNA. The single-strandedsiRNAs are generally 15-30 nucleotides and are chemically modified. Thedesign and testing of single-stranded siRNAs are described in U.S. Pat.No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In certain embodiments, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double stranded RNA and is referred toherein as a “double stranded RNA agent,” “double stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., an AGT gene. In some embodiments ofthe invention, a double stranded RNA (dsRNA) triggers the degradation ofa target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide or a modified nucleotide. In addition, as used inthis specification, an “iRNA” may include ribonucleotides with chemicalmodifications; an iRNA may include substantial modifications at multiplenucleotides. As used herein, the term “modified nucleotide” refers to anucleotide having, independently, a modified sugar moiety, a modifiedinternucleotide linkage, or modified nucleobase, or any combinationthereof. Thus, the term modified nucleotide encompasses substitutions,additions or removal of, e.g., a functional group or atom, tointernucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“iRNA” or “RNAi agent” for the purposes of this specification andclaims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about19 to 36 base pairs in length, e.g., about 19-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25,19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26,20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26,21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengthsintermediate to the above recited ranges and lengths are alsocontemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments,the hairpin loop can be 10 or fewer nucleotides. In some embodiments,the hairpin loop can be 8 or fewer unpaired nucleotides. In someembodiments, the hairpin loop can be 4-10 unpaired nucleotides. In someembodiments, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not be, butcan be covalently connected. Where the two strands are connectedcovalently by means other than an uninterrupted chain of nucleotidesbetween the 3′-end of one strand and the 5′-end of the respective otherstrand forming the duplex structure, the connecting structure isreferred to as a “linker.” The RNA strands may have the same or adifferent number of nucleotides. The maximum number of base pairs is thenumber of nucleotides in the shortest strand of the dsRNA minus anyoverhangs that are present in the duplex. In addition to the duplexstructure, an RNAi may comprise one or more nucleotide overhangs.

In certain embodiments, an iRNA agent of the invention is a dsRNA, eachstrand of which comprises 19-23 nucleotides, that interacts with atarget RNA sequence, e.g., an AGT gene, to direct cleavage of the targetRNA.

In some embodiments, an iRNA of the invention is a dsRNA of 24-30nucleotides that interacts with a target RNA sequence, e.g., an AGTtarget mRNA sequence, to direct the cleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of a doublestranded iRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand, or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5-end, 3′-end, orboth ends of either an antisense or sense strand of a dsRNA.

In certain embodiments, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end or the 5′-end. In certain embodiments, theoverhang on the sense strand or the antisense strand, or both, caninclude extended lengths longer than 10 nucleotides, e.g., 1-30nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides,10-20 nucleotides, or 10-15 nucleotides in length. In certainembodiments, an extended overhang is on the sense strand of the duplex.In certain embodiments, an extended overhang is present on the 3′ end ofthe sense strand of the duplex. In certain embodiments, an extendedoverhang is present on the 5′ end of the sense strand of the duplex. Incertain embodiments, an extended overhang is on the antisense strand ofthe duplex. In certain embodiments, an extended overhang is present onthe 3′ end of the antisense strand of the duplex. In certainembodiments, an extended overhang is present on the 5′ end of theantisense strand of the duplex. In certain embodiments, one or more ofthe nucleotides in the extended overhang is replaced with a nucleosidethiophosphate. In certain embodiments, the overhang includes aself-complementary portion such that the overhang is capable of forminga hairpin structure that is stable under physiological conditions.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double stranded RNA agent, i.e., no nucleotide overhang.A “blunt ended” double stranded RNA agent is double stranded over itsentire length, i.e., no nucleotide overhang at either end of themolecule. The RNAi agents of the invention include RNAi agents with nonucleotide overhang at one end (i.e., agents with one overhang and oneblunt end) or with no nucleotide overhangs at either end. Most oftensuch a molecule will be double-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., an AGT mRNA. As used herein,the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., an AGT nucleotide sequence, as definedherein. Where the region of complementarity is not fully complementaryto the target sequence, the mismatches can be in the internal orterminal regions of the molecule. Generally, the most toleratedmismatches are in the terminal regions, e.g., within 5, 4, or 3nucleotides of the 5′- or 3′-end of the iRNA. In some embodiments, adouble stranded RNA agent of the invention includes a nucleotidemismatch in the antisense strand. In some embodiments, a double strandedRNA agent of the invention includes a nucleotide mismatch in the sensestrand. In some embodiments, the nucleotide mismatch is, for example,within 5, 4, 3 nucleotides from the 3′-end of the iRNA. In anotherembodiment, the nucleotide mismatch is, for example, in the 3′-terminalnucleotide of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, “substantially all of the nucleotides are modified” arelargely but not wholly modified and can include not more than 5, 4, 3,2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3, or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs or base pairs formedfrom non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a double stranded RNA agent and a targetsequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding an AGT gene). For example, apolynucleotide is complementary to at least a part of an AGT mRNA if thesequence is substantially complementary to a non-interrupted portion ofan mRNA encoding an AGT gene.

Accordingly, in some embodiments, the sense strand polynucleotides andthe antisense polynucleotides disclosed herein are fully complementaryto the target AGT sequence. In other embodiments, the sense strandpolynucleotides or the antisense polynucleotides disclosed herein aresubstantially complementary to the target AGT sequence and comprise acontiguous nucleotide sequence which is at least 80% complementary overits entire length to the equivalent region of the nucleotide sequence ofany one of SEQ ID NOs:1 and 2, or a fragment of any one of SEQ ID NOs:1and 2, such as at least 90%, or 95% complementary; or 100%complementary.

Accordingly, in some embodiments, the antisense strand polynucleotidesdisclosed herein are fully complementary to the target AGT sequence. Inother embodiments, the antisense strand polynucleotides disclosed hereinare substantially complementary to the target AGT sequence and comprisea contiguous nucleotide sequence which is at least about 90%complementary over its entire length to the equivalent region of thenucleotide sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1, suchas about 90%, or about 95%, complementary. In certain embodiments, thefragment of SEQ ID NO: 1 is selected from the group of nucleotides632-658, 635-658, 636-658, 1248-1273, 1248-1270, 1250-1272, 1251-1273,1580-1602, 1584-1606, 1587-1609, 1601-1623, 1881-1903, 2074-2097,2074-2096, 2075-2097, 2080-2102, 2272-2294, 2276-2298, 2281-2304,2281-2303, or 2282-2304 of SEQ ID NO: 1. In preferred embodiments, theduplex does not consist of the sense strand consisting ofuscsucccAfcCfUfUfuucuucuaauL96 (SEQ ID NO: 13) and the antisense strandconsisting of asUfsuagAfagaaaagGfuGfggagascsu (SEQ ID NO: 14).

In some embodiments, an iRNA of the invention includes an antisensestrand that is substantially complementary to the target AGT sequenceand comprises a contiguous nucleotide sequence which is at least about90% complementary over its entire length to the equivalent region of thenucleotide sequence of any one of the sense strands in Table 3, Table 5,or Table 6 or a fragment of any one of the sense strands in Table 3,Table 5, or Table 6, such as about 90%, 95%, or 100% complementary.

In some embodiments, an iRNA of the invention includes a sense strandthat is substantially complementary to an antisense polynucleotidewhich, in turn, is complementary to a target AGT sequence, and whereinthe sense strand polynucleotide comprises a contiguous nucleotidesequence which is at least about 90% complementary over its entirelength to the equivalent region of the nucleotide sequence of any one ofthe antisense strands in Table 3, 5, or 6, or a fragment of any one ofthe antisense strands in Table 3 or 5, such as about 90%, 95%, or 100%.

In certain embodiments, the sense and antisense strands in Table 3 orTable 5 are selected from duplexes AD-85481, AD-84701, AD-84703,AD-84704, AD-84705, AD-84707, AD-84715, AD-84716, AD-84739, AD-84741,AD-84746, AD-85432, AD-85434, AD-85435, AD-85436, AD-85437, AD-85438,AD-85441, AD-85442, AD-85443, AD-85444, AD-85446, AD-85447, AD-85482,AD-85485, AD-85493, AD-85496, AD-85504, AD-85517, AD-85519, AD-85524,AD-85622, AD-85623, AD-85625, AD-85626, AD-85634, AD-85635, AD-85637,and AD-85655.

In general, an “iRNA” includes ribonucleotides with chemicalmodifications. Such modifications may include all types of modificationsdisclosed herein or known in the art. Any such modifications, as used ina dsRNA molecule, are encompassed by “iRNA” for the purposes of thisspecification and claims.

In an aspect of the invention, an agent for use in the methods andcompositions of the invention is a single-stranded antisenseoligonucleotide molecule that inhibits a target mRNA via an antisenseinhibition mechanism. The single-stranded antisense oligonucleotidemolecule is complementary to a sequence within the target mRNA. Thesingle-stranded antisense oligonucleotides can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355. The single-stranded antisense oligonucleotidemolecule may be about 14 to about 30 nucleotides in length and have asequence that is complementary to a target sequence. For example, thesingle-stranded antisense oligonucleotide molecule may comprise asequence that is at least about 14, 15, 16, 17, 18, 19, 20, or morecontiguous nucleotides from any one of the antisense sequences describedherein.

The phrase “contacting a cell with an iRNA,” such as a dsRNA, as usedherein, includes contacting a cell by any possible means. Contacting acell with an iRNA includes contacting a cell in vitro with the iRNA orcontacting a cell in vivo with the iRNA. The contacting may be donedirectly or indirectly. Thus, for example, the iRNA may be put intophysical contact with the cell by the individual performing the method,or alternatively, the iRNA may be put into a situation that will permitor cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the iRNA. Contacting a cell in vivo may be done, for example,by injecting the iRNA into or near the tissue where the cell is located,or by injecting the iRNA into another area, e.g., the bloodstream or thesubcutaneous space, such that the agent will subsequently reach thetissue where the cell to be contacted is located. For example, the iRNAmay contain or be coupled to a ligand, e.g., GaNAc, that directs theiRNA to a site of interest, e.g., the liver. Combinations of in vitroand in vivo methods of contacting are also possible. For example, a cellmay also be contacted in vitro with an iRNA and subsequentlytransplanted into a subject.

In certain embodiments, contacting a cell with an iRNA includes“introducing” or “delivering the iRNA into the cell” by facilitating oreffecting uptake or absorption into the cell. Absorption or uptake of aniRNA can occur through unaided diffusion or active cellular processes,or by auxiliary agents or devices. Introducing an iRNA into a cell maybe in vitro or in vivo. For example, for in vivo introduction, iRNA canbe injected into a tissue site or administered systemically. In vitrointroduction into a cell includes methods known in the art such aselectroporation and lipofection. Further approaches are described hereinbelow or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a horse, a goat, arabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or amouse), or a bird that expresses the target gene, either endogenously orheterologously. In an embodiment, the subject is a human, such as ahuman being treated or assessed for a disease or disorder that wouldbenefit from reduction in AGT expression; a human at risk for a diseaseor disorder that would benefit from reduction in AGT expression; a humanhaving a disease or disorder that would benefit from reduction in AGTexpression; or human being treated for a disease or disorder that wouldbenefit from reduction in AGT expression as described herein. Thediagnostic criteria for an AGT-associated disorder, e.g., hypertension,are provided below. In some embodiments, the subject is a female human.In other embodiments, the subject is a male human. In certainembodiments, the subject is part of a group susceptible to saltsensitivity, e.g., black or an older adult (>65 years of age). Incertain embodiments, the subject is overweight or obese, e.g., a subjectthat suffers from central obesity. In certain embodiments, the subjectis sedentary. In certain embodiments, the subject is pregnant.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result, such as reducing at least one sign orsymptom of an AGT-associated disorder, e.g., hypertension in a subject.Treatment also includes a reduction of one or more sign or symptomsassociated with unwanted AGT expression, e.g., angiotensin II type 1receptor activation (AT₁R) (e.g., hypertension, chronic kidney disease,stroke, myocardial infarction, heart failure, aneurysms, peripheralartery disease, heart disease, increased oxidative stress, e.g.,increased superoxide formation, inflammation, vasoconstriction, sodiumand water retention, potassium and magnesium loss, renin suppression,myocyte and smooth muscle hypertrophy, increased collagen synthesis,stimulation of vascular, myocardial and renal fibrosis, increased rateand force of cardiac contractions, altered heart rate, e.g., increasedarrhythmia, stimulation of plasminogen activator inhibitor 1 (PAI1),activation of the sympathetic nervous system, and increased endothelinsecretion), symptoms of pregnancy-associated hypertension (e.g.,preeclampsia, and eclampsia), including, but not limited to intrauterinegrowth restriction (IUGR) or fetal growth restriction, symptomsassociated with malignant hypertension, symptoms associated withhyperaldosteronism; diminishing the extent of unwanted AT₁R activation;stabilization (i.e., not worsening) of the state of chronic AT₁Ractivation; amelioration or palliation of unwanted AT₁R activation(e.g., hypertension, chronic kidney disease, stroke, myocardialinfarction, heart failure, aneurysms, peripheral artery disease, heartdisease, increased oxidative stress, e.g., increased superoxideformation, inflammation, vasoconstriction, sodium and water retention,potassium and magnesium loss, renin suppression, myocyte and smoothmuscle hypertrophy, increased collagen synthesis, stimulation ofvascular, myocardial and renal fibrosis, increased rate and force ofcardiac contractions, altered heart rate, e.g., increased arrhythmia,stimulation of plasminogen activator inhibitor 1 (PAI1), activation ofthe sympathetic nervous system, and increased endothelin secretion)whether detectable or undetectable. AGT-associated disorders can alsoinclude obesity, liver steatosis/fatty liver, e.g., non-alcoholicSteatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD),glucose intolerance, type 2 diabetes (non-insulin dependent diabetes),and metabolic syndrome. In certain embodiments, hypertension includeshypertension associated with low plasma renin activity or plasma reninconcentration. “Treatment” can also mean prolonging survival as comparedto expected survival in the absence of treatment.

The term “lower” in the context of the level of AGT gene expression oragt protein production in a subject, or a disease marker or symptomrefers to a statistically significant decrease in such level. Thedecrease can be, for example, at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95%, or below the level of detection for the detectionmethod in a relevant cell or tissue, e.g., a liver cell, or othersubject sample, e.g., blood or serum derived therefrom, urine.

As used herein, “prevention” or “preventing,” when used in reference toa disease or disorder, that would benefit from a reduction in expressionof an AGT gene or production of agt protein, e.g., in a subjectsusceptible to an AGT-associated disorder due to, e.g., aging, geneticfactors, hormone changes, diet, and a sedentary lifestyle. In certainembodiments, the disease or disorder is e.g., a symptom of unwanted AT₁Ractivation, such as a hypertension, chronic kidney disease, stroke,myocardial infarction, heart failure, aneurysms, peripheral arterydisease, heart disease, increased oxidative stress, e.g., increasedsuperoxide formation, inflammation, vasoconstriction, sodium and waterretention, potassium and magnesium loss, renin suppression, myocyte andsmooth muscle hypertrophy, increased collagen synthesis, stimulation ofvascular, myocardial and renal fibrosis, increased rate and force ofcardiac contractions, altered heart rate, e.g., increased arrhythmia,stimulation of plasminogen activator inhibitor 1 (PAI1), activation ofthe sympathetic nervous system, and increased endothelin secretion.AGT-associated disorders can also include obesity, liver steatosis/fattyliver, e.g., non-alcoholic Steatohepatitis (NASH) and non-alcoholicfatty liver disease (NAFLD), glucose intolerance, type 2 diabetes(non-insulin dependent diabetes), and metabolic syndrome. In certainembodiments, hypertension includes hypertension associated with lowplasma renin activity or plasma renin concentration. The likelihood ofdeveloping, e.g., hypertension, is reduced, for example, when anindividual having one or more risk factors for a hypertension eitherfails to develop hypertension or develops hypertension with lessseverity relative to a population having the same risk factors and notreceiving treatment as described herein. The failure to develop anAGT-associated disorder, e.g., hypertension or a delay in the time todevelop hypertension by months or years is considered effectiveprevention. Prevention may require administration of more than one doseif the iRNA agent.

As used herein, the term “angiotensinogen-associated disease” or“AGT-associated disease,” is a disease or disorder that is caused by, orassociated with renin-angiotensin-aldosterone system (RAAS) activation,or a disease or disorder the symptoms of which or progression of whichresponds to RAAS inactivation. The term “angiotensinogen-associateddisease” includes a disease, disorder or condition that would benefitfrom reduction in AGT expression. Such diseases are typically associatedwith high blood pressure. Non-limiting examples ofangiotensinogen-associated diseases include hypertension, e.g.,borderline hypertension (also known as prehypertension), primaryhypertension (also known as essential hypertension or idiopathichypertension), secondary hypertension (also known as inessentialhypertension), isolated systolic or diastolic hypertension,pregnancy-associated hypertension (e.g., preeclampsia, eclampsia, andpost-partum preelampsia), diabetic hypertension, resistant hypertension,refractory hypertension, paroxysmal hypertension, renovascularhypertension (also known as renal hypertension), Goldblatt hypertension,ocular hypertension, glaucoma, pulmonary hypertension, portalhypertension, systemic venous hypertension, systolic hypertension,labile hypertension; hypertensive heart disease, hypertensivenephropathy, atherosclerosis, arteriosclerosis, vasculopathy (includingperipheral vascular disease), diabetic nephropathy, diabeticretinopathy, chronic heart failure, cardiomyopathy, diabetic cardiacmyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurism,ventricular fibrosis, sleep apnea, heart failure (e.g., left ventricularsystolic dysfunction), myocardial infarction, angina, stroke, renaldisease e.g., chronic kidney disease or diabetic nephropathy optionallyin the context of pregnancy, renal failure, e.g., chronic renal failure,and systemic sclerosis (e.g., scleroderma renal crisis). In certainembodiments, AGT-associated disease includes intrauterine growthrestriction (IUGR) or fetal growth restriction. In certain embodiments,AGT-associated disorders also include obesity, liver steatosis/fattyliver, e.g., non-alcoholic Steatohepatitis (NASH) and non-alcoholicfatty liver disease (NAFLD), glucose intolerance, type 2 diabetes(non-insulin dependent diabetes), and metabolic syndrome. In certainembodiments, hypertension includes hypertension associated with lowplasma renin activity or plasma renin concentration.

Thresholds for high blood pressure and stages of hypertension arediscussed in detail below.

In one embodiment, an angiotensinogen-associated disease is primaryhypertension. “Primary hypertension” is a result of environmental orgenetic causes (e.g., a result of no obvious underlying medical cause).

In one embodiment, an angiotensinogen-associated disease is secondaryhypertension. “Secondary hypertension” has an identifiable underlyingdisorder which can be of multiple etiologies, including renal, vascular,and endocrine causes, e.g., renal parenchymal disease (e.g., polycystickidneys, glomerular or interstitial disease), renal vascular disease(e.g., renal artery stenosis, fibromuscular dysplasia), endocrinedisorders (e.g., adrenocorticosteroid or mineralocorticoid excess,pheochromocytoma, hyperthyroidism or hypothyroidism, growth hormoneexcess, hyperparathyroidism), coarctation of the aorta, or oralcontraceptive use.

In one embodiment, an angiotensinogen-associated disease ispregnancy-associated hypertension, e.g., chronic hypertension ofpregnancy, gestational hypertension, preeclampsia, eclampsia,preeclampsia superimposed on chronic hypertension, HELLP syndrome, andgestational hypertension (also known as transient hypertension ofpregnancy, chronic hypertension identified in the latter half ofpregnancy, and pregnancy-induced hypertension (PIH)). Diagnosticcriteria for pregnancy-associated hypertension are provided below.

In one embodiment, an angiotensinogen-associated disease is resistanthypertension. “Resistant hypertension” is blood pressure that remainsabove goal (e.g., above 130 mm Hg systolic or above 90 diastolic) inspite of concurrent use of three antihypertensive agents of differentclasses, one of which is a thiazide diuretic. Subjects whose bloodpressure is controlled with four or more medications are also consideredto have resistant hypertension.

A “therapeutically-effective amount” or “prophylactically effectiveamount” also includes an amount of an RNAi agent that produces somedesired effect at a reasonable benefit/risk ratio applicable to anytreatment. The iRNA employed in the methods of the present invention maybe administered in a sufficient amount to produce a reasonablebenefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Such carriers are knownin the art. Pharmaceutically acceptable carriers include carriers foradministration by injection.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs, or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In some embodiments, a “sample derived from a subject”refers to urine obtained from the subject. A “sample derived from asubject” can refer to blood or blood derived serum or plasma from thesubject.

I. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of anAGT gene. In preferred embodiments, the iRNA includes double strandedribonucleic acid (dsRNA) molecules for inhibiting the expression of anAGT gene in a cell, such as a cell within a subject, e.g., a mammal,such as a human susceptible to developing an AGT-associated disorder,e.g., hypertension. The dsRNAi agent includes an antisense strand havinga region of complementarity which is complementary to at least a part ofan mRNA formed in the expression of an AGT gene. The region ofcomplementarity is about 19-30 nucleotides in length (e.g., about 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).Upon contact with a cell expressing the AGT gene, the iRNA inhibits theexpression of the AGT gene (e.g., a human, a primate, a non-primate, ora rat AGT gene) by at least about 50% as assayed by, for example, a PCRor branched DNA (bDNA)-based method, or by a protein-based method, suchas by immunofluorescence analysis, using, for example, western blottingor flow cytometric techniques. In preferred embodiments, inhibition ofexpression is determined by the qPCR method provided in the examples,especially in Example 2 with the siRNA at a 10 nM concentration in anappropriate organism cell line provided therein. In preferredembodiments, inhibition of expression in vivo is determined by knockdownof the human gene in a rodent expressing the human gene, e.g., a mouseor an AAV-infected mouse expressing the human target gene, e.g., whenadministered a single dose at 3 mg/kg at the nadir of RNA expression.RNA expression in liver is determined using the PCR methods provided inExample 2.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of an AGTgene. The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is 19 to 30 base pairs in length.Similarly, the region of complementarity to the target sequence is 19 to30 nucleotides in length.

In some embodiments, the dsRNA is about 19 to about 23 nucleotides inlength, or about 25 to about 30 nucleotides in length. In general, thedsRNA is long enough to serve as a substrate for the Dicer enzyme. Forexample, it is well-known in the art that dsRNAs longer than about 21-23nucleotides in length may serve as substrates for Dicer. As theordinarily skilled person will also recognize, the region of an RNAtargeted for cleavage will most often be part of a larger RNA molecule,often an mRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to allow itto be a substrate for RNAi-directed cleavage (i.e., cleavage through aRISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 19to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in oneembodiment, to the extent that it becomes processed to a functionalduplex, of e.g., 15-30 base pairs, that targets a desired RNA forcleavage, an RNA molecule or complex of RNA molecules having a duplexregion greater than 30 base pairs is a dsRNA. Thus, an ordinarilyskilled artisan will recognize that in one embodiment, a miRNA is adsRNA. In another embodiment, a dsRNA is not a naturally occurringmiRNA. In another embodiment, an iRNA agent useful to target AGT geneexpression is not generated in the target cell by cleavage of a largerdsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3,or 4 nucleotides. dsRNAs having at least one nucleotide overhang canhave superior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand, or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end, or both ends of an antisense or sensestrand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art. Doublestranded RNAi compounds of the invention may be prepared using atwo-step procedure. First, the individual strands of the double strandedRNA molecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Similarly, single-stranded oligonucleotides of the invention can beprepared using solution-phase or solid-phase organic synthesis or both.

In an aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandis selected from the group of sequences provided in Tables 3, 5, and 6,and the corresponding antisense strand of the sense strand is selectedfrom the group of sequences of Table 3, 5, and 6. In this aspect, one ofthe two sequences is complementary to the other of the two sequences,with one of the sequences being substantially complementary to asequence of an mRNA generated in the expression of an AGT gene. As such,in this aspect, a dsRNA will include two oligonucleotides, where oneoligonucleotide is described as the sense strand in Table, 5, or 6, andthe second oligonucleotide is described as the corresponding antisensestrand of the sense strand in Table 3, 5, or 6. In certain embodiments,the substantially complementary sequences of the dsRNA are contained onseparate oligonucleotides. In other embodiments, the substantiallycomplementary sequences of the dsRNA are contained on a singleoligonucleotide. In certain embodiments, the sense or antisense strandfrom Table 3 or 5 is selected from AD-85481, AD-84701, AD-84703,AD-84704, AD-84705, AD-84707, AD-84715, AD-84716, AD-84739, AD-84741,AD-84746, AD-85432, AD-85434, AD-85435, AD-85436, AD-85437, AD-85438,AD-85441, AD-85442, AD-85443, AD-85444, AD-85446, AD-85447, AD-85482,AD-85485, AD-85493, AD-85496, AD-85504, AD-85517, AD-85519, AD-85524,AD-85622, AD-85623, AD-85625, AD-85626, AD-85634, AD-85635, AD-85637,and AD-85655.

It will be understood that, although the sequences in Table 3 are notdescribed as modified or conjugated sequences, the RNA of the iRNA ofthe invention e.g., a dsRNA of the invention, may comprise any one ofthe sequences set forth in Table 3, or the sequences of Table 5 or 6that are modified, or the sequences of Table 5 or 6 that are conjugated.In other words, the invention encompasses dsRNA of Table 3, 5, and 6which are un-modified, un-conjugated, modified, or conjugated, asdescribed herein.

The skilled person is well aware that dsRNAs having a duplex structureof about 20 to 23 base pairs, e.g., 21, base pairs have been hailed asparticularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger RNA duplex structures can also be effective (Chu and Rana (2007)RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Table 3, 5, and 6,dsRNAs described herein can include at least one strand of a length ofminimally 21 nucleotides. It can be reasonably expected that shorterduplexes having one of the sequences of Table 3, 5, and 6 minus only afew nucleotides on one or both ends can be similarly effective ascompared to the dsRNAs described above. Hence, dsRNAs having a sequenceof at least 19, 20, or more contiguous nucleotides derived from one ofthe sequences of Table 3, 5, and 6, and differing in their ability toinhibit the expression of an AGT gene by not more than about 5, 10, 15,20, 25, or 30% inhibition from a dsRNA comprising the full sequence, arecontemplated to be within the scope of the present invention.

In addition, the RNAs provided in Table 3, 5, and 6 identify a site(s)in an AGT transcript that is susceptible to RISC-mediated cleavage. Assuch, the present invention further features iRNAs that target withinone of these sites. As used herein, an iRNA is said to target within aparticular site of an RNA transcript if the iRNA promotes cleavage ofthe transcript anywhere within that particular site. Such an iRNA willgenerally include at least about 19 contiguous nucleotides from one ofthe sequences provided in Table 3, 5, and 6 coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in an AGT gene.

II. Modified iRNAs of the Invention

In certain embodiments, the RNA of the iRNA of the invention e.g., adsRNA, is un-modified, and does not comprise, e.g., chemicalmodifications or conjugations known in the art and described herein. Inother embodiments, the RNA of an iRNA of the invention, e.g., a dsRNA,is chemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA orsubstantially all of the nucleotides of an iRNA are modified, i.e., notmore than 5, 4, 3, 2, or 1 unmodified nucleotides are present in astrand of the iRNA.

The nucleic acids featured in the invention can be synthesized ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; or backbonemodifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. Patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts.

Representative U.S. Patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

Suitable RNA mimetics are contemplated for use in iRNAs provided herein,in which both the sugar and the internucleoside linkage, i.e., thebackbone, of the nucleotide units are replaced with novel groups. Thebase units are maintained for hybridization with an appropriate nucleicacid target compound. One such oligomeric compound in which an RNAmimetic that has been shown to have excellent hybridization propertiesis referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative US patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents ofeach of which are hereby incorporated herein by reference.

Additional PNA compounds suitable for use in the iRNAs of the inventionare described in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂) CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)CH₃)]2, where n and m are from 1to about 10. In other embodiments, dsRNAs include one of the followingat the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an iRNA, or a group forimproving the pharmacodynamic properties of an iRNA, and othersubstituents having similar properties. In some embodiments, themodification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂CH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modificationis 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also knownas 2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include:5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides,5′-Me-2′-deoxynucleotides, (both R and S isomers in these threefamilies); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative US patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C), anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. Patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

In some embodiments, the RNA of an iRNA can also be modified to includeone or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosylring modified by the bridging of two atoms. A “bicyclic nucleoside”(“BNA”) is a nucleoside having a sugar moiety comprising a bridgeconnecting two carbon atoms of the sugar ring, thereby forming abicyclic ring system. In certain embodiments, the bridge connects the4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodimentsan agent of the invention may include one or more locked nucleic acids(LNA). A locked nucleic acid is a nucleotide having a modified ribosemoiety in which the ribose moiety comprises an extra bridge connectingthe 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprisinga bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. This structureeffectively “locks” the ribose in the 3′-endo structural conformation.The addition of locked nucleic acids to siRNAs has been shown toincrease siRNA stability in serum, and to reduce off-target effects(Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al.,(2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclicnucleosides for use in the polynucleotides of the invention includewithout limitation nucleosides comprising a bridge between the 4′ andthe 2′ ribosyl ring atoms. In certain embodiments, the antisensepolynucleotide agents of the invention include one or more bicyclicnucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′bridged bicyclic nucleosides, include but are not limited to4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ (also referred to as “constrained ethyl” or “cEt”) and4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof, see, e.g., U.S. Pat. No.7,399,845); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof; see e.g., U.S.Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof, see e.g.,U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., U.S. PatentPublication No. 2004/0171570); 4′-CH₂—N(R)—O-2′, wherein R is H, C1-C12alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672);4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem.,2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof, see,e.g., U.S. Pat. No. 8,278,426). The entire contents of each of theforegoing are hereby incorporated herein by reference.

Additional representative U.S. Patents and U.S. Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, U.S. Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

In some embodiments, an iRNA of the invention comprises one or moremonomers that are UNA (unlocked nucleic acid) nucleotides. UNA isunlocked acyclic nucleic acid, wherein any of the bonds of the sugar hasbeen removed, forming an unlocked “sugar” residue. In one example, UNAalso encompasses monomer with bonds between C1′-C4′ have been removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar hasbeen removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) andFluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated byreference).

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and U.S.Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020,the entire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of the nucleotides of an iRNA of the inventioninclude a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminalphosphate or phosphate mimic on the antisense strand of an iRNA.Suitable phosphate mimics are disclosed in, for example U.S. PatentPublication No. 2012/0157511, the entire contents of which areincorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNA agents ofthe invention include agents with chemical modifications as disclosed,for example, in WO2013/075035, the entire contents of each of which areincorporated herein by reference. WO2013/075035 provides motifs of threeidentical modifications on three consecutive nucleotides into a sensestrand or antisense strand of a dsRNAi agent, particularly at or nearthe cleavage site. In some embodiments, the sense strand and antisensestrand of the dsRNAi agent may otherwise be completely modified. Theintroduction of these motifs interrupts the modification pattern, ifpresent, of the sense or antisense strand. The dsRNAi agent may beoptionally conjugated with a GalNAc derivative ligand, for instance onthe sense strand.

More specifically, when the sense strand and antisense strand of thedouble stranded RNA agent are completely modified to have one or moremotifs of three identical modifications on three consecutive nucleotidesat or near the cleavage site of at least one strand of a dsRNAi agent,the gene silencing activity of the dsRNAi agent was observed.

Accordingly, the invention provides double stranded RNA agents capableof inhibiting the expression of a target gene (i.e., AGT gene) in vivo.The RNAi agent comprises a sense strand and an antisense strand. Eachstrand of the RNAi agent may be, for example, 17-30 nucleotides inlength, 25-30 nucleotides in length, 27-30 nucleotides in length, 19-25nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides inlength, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as “dsRNAi agent.” Theduplex region of a dsRNAi agent may be, for example, the duplex regioncan be 27-30 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 19, 20,21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In certain embodiments, the dsRNAi agent may contain one or moreoverhang regions or capping groups at the 3′-end, 5′-end, or both endsof one or both strands. The overhang can be, independently, 1-6nucleotides in length, for instance 2-6 nucleotides in length, 1-5nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides inlength, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3nucleotides in length, or 1-2 nucleotides in length. In certainembodiments, the overhang regions can include extended overhang regionsas provided above. The overhangs can be the result of one strand beinglonger than the other, or the result of two strands of the same lengthbeing staggered. The overhang can form a mismatch with the target mRNAor it can be complementary to the gene sequences being targeted or canbe another sequence. The first and second strands can also be joined,e.g., by additional bases to form a hairpin, or by other non-baselinkers.

In certain embodiments, the nucleotides in the overhang region of thedsRNAi agent can each independently be a modified or unmodifiednucleotide including, but no limited to 2′-sugar modified, such as,2′-F, 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine(Teo), 2′-O-methoxyethyladenosine (Aeo),2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinationsthereof. For example, TT can be an overhang sequence for either end oneither strand. The overhang can form a mismatch with the target mRNA orit can be complementary to the gene sequences being targeted or can beanother sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand, or bothstrands of the dsRNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In some embodiments, the overhang ispresent at the 3′-end of the sense strand, antisense strand, or bothstrands. In some embodiments, this 3′-overhang is present in theantisense strand. In some embodiments, this 3′-overhang is present inthe sense strand.

The dsRNAi agent may contain only a single overhang, which canstrengthen the interference activity of the RNAi, without affecting itsoverall stability. For example, the single-stranded overhang may belocated at the 3′-end of the sense strand or, alternatively, at the3′-end of the antisense strand. The RNAi may also have a blunt end,located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of thedsRNAi agent has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. While not wishing to be bound by theory, the asymmetric blunt endat the 5′-end of the antisense strand and 3′-end overhang of theantisense strand favor the guide strand loading into RISC process.

In certain embodiments, the dsRNAi agent is a double ended bluntmer of19 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In other embodiments, the dsRNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In yet other embodiments, the dsRNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In certain embodiments, the dsRNAi agent comprises a 21 nucleotide sensestrand and a 23 nucleotide antisense strand, wherein the sense strandcontains at least one motif of three 2′-F modifications on threeconsecutive nucleotides at positions 9, 10, 11 from the 5′ end; theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at positions 11, 12, 13from the 5′ end, wherein one end of the RNAi agent is blunt, while theother end comprises a 2 nucleotide overhang. Preferably, the 2nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand,there may be two phosphorothioate internucleotide linkages between theterminal three nucleotides, wherein two of the three nucleotides are theoverhang nucleotides, and the third nucleotide is a paired nucleotidenext to the overhang nucleotide. In one embodiment, the RNAi agentadditionally has two phosphorothioate internucleotide linkages betweenthe terminal three nucleotides at both the 5′-end of the sense strandand at the 5′-end of the antisense strand. In certain embodiments, everynucleotide in the sense strand and the antisense strand of the dsRNAiagent, including the nucleotides that are part of the motifs aremodified nucleotides. In certain embodiments each residue isindependently modified with a 2′-O-methyl or 3′-fluoro, e.g., in analternating motif Optionally, the dsRNAi agent further comprises aligand (preferably GalNAc₃).

In certain embodiments, the dsRNAi agent comprises a sense and anantisense strand, wherein the sense strand is 25-30 nucleotide residuesin length, wherein starting from the 5′ terminal nucleotide (position 1)positions 1 to 23 of the first strand comprise at least 8ribonucleotides; the antisense strand is 36-66 nucleotide residues inlength and, starting from the 3′ terminal nucleotide, comprises at least8 ribonucleotides in the positions paired with positions 1-23 of sensestrand to form a duplex; wherein at least the 3′ terminal nucleotide ofantisense strand is unpaired with sense strand, and up to 6 consecutive3′ terminal nucleotides are unpaired with sense strand, thereby forminga 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′terminus of antisense strand comprises from 10-30 consecutivenucleotides which are unpaired with sense strand, thereby forming a10-30 nucleotide single stranded 5′ overhang; wherein at least the sensestrand 5′ terminal and 3′ terminal nucleotides are base paired withnucleotides of antisense strand when sense and antisense strands arealigned for maximum complementarity, thereby forming a substantiallyduplexed region between sense and antisense strands; and antisensestrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of antisense strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell; and wherein the sense strand contains at least one motifof three 2′-F modifications on three consecutive nucleotides, where atleast one of the motifs occurs at or near the cleavage site. Theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at or near the cleavagesite.

In certain embodiments, the dsRNAi agent comprises sense and antisensestrands, wherein the dsRNAi agent comprises a first strand having alength which is at least 25 and at most 29 nucleotides and a secondstrand having a length which is at most 30 nucleotides with at least onemotif of three 2′-O-methyl modifications on three consecutivenucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ endof the first strand and the 5′ end of the second strand form a blunt endand the second strand is 1-4 nucleotides longer at its 3′ end than thefirst strand, wherein the duplex region which is at least 25 nucleotidesin length, and the second strand is sufficiently complementary to atarget mRNA along at least 19 nucleotide of the second strand length toreduce target gene expression when the RNAi agent is introduced into amammalian cell, and wherein Dicer cleavage of the dsRNAi agentpreferentially results in an siRNA comprising the 3′-end of the secondstrand, thereby reducing expression of the target gene in the mammal.Optionally, the dsRNAi agent further comprises a ligand.

In certain embodiments, the sense strand of the dsRNAi agent contains atleast one motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In certain embodiments, the antisense strand of the dsRNAi agent canalso contain at least one motif of three identical modifications onthree consecutive nucleotides, where one of the motifs occurs at or nearthe cleavage site in the antisense strand.

For a dsRNAi agent having a duplex region of 19-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10,11, and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; the 10, 11, 12positions; the 11, 12, 13 positions; the 12, 13, 14 positions; or the13, 14, 15 positions of the antisense strand, the count starting fromthe first nucleotide from the 5′-end of the antisense strand, or, thecount starting from the first paired nucleotide within the duplex regionfrom the 5′-end of the antisense strand. The cleavage site in theantisense strand may also change according to the length of the duplexregion of the dsRNAi agent from the 5′-end.

The sense strand of the dsRNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In some embodiments, the sense strand of the dsRNAi agent may containmore than one motif of three identical modifications on threeconsecutive nucleotides. The first motif may occur at or near thecleavage site of the strand and the other motifs may be a wingmodification. The term “wing modification” herein refers to a motifoccurring at another portion of the strand that is separated from themotif at or near the cleavage site of the same strand. The wingmodification is either adjacent to the first motif or is separated by atleast one or more nucleotides. When the motifs are immediately adjacentto each other then the chemistries of the motifs are distinct from eachother, and when the motifs are separated by one or more nucleotide thanthe chemistries can be the same or different. Two or more wingmodifications may be present. For instance, when two wing modificationsare present, each wing modification may occur at one end relative to thefirst motif which is at or near cleavage site or on either side of thelead motif.

Like the sense strand, the antisense strand of the dsRNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In some embodiments, the wing modification on the sense strand orantisense strand of the dsRNAi agent typically does not include thefirst one or two terminal nucleotides at the 3′-end, 5′-end, or bothends of the strand.

In other embodiments, the wing modification on the sense strand orantisense strand of the dsRNAi agent typically does not include thefirst one or two paired nucleotides within the duplex region at the3′-end, 5′-end, or both ends of the strand.

When the sense strand and the antisense strand of the dsRNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two,or three nucleotides.

When the sense strand and the antisense strand of the dsRNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two, or three nucleotides; two modifications each from one strand fallon the other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In some embodiments, every nucleotide in the sense strand and antisensestrand of the dsRNAi agent, including the nucleotides that are part ofthe motifs, may be modified. Each nucleotide may be modified with thesame or different modification which can include one or more alterationof one or both of the non-linking phosphate oxygens or of one or more ofthe linking phosphate oxygens; alteration of a constituent of the ribosesugar, e.g., of the 2′-hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′- or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of an RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′-end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′- or 3′-overhang,or in both. For example, it can be desirable to include purinenucleotides in overhangs. In some embodiments all or some of the basesin a 3′- or 5′-overhang may be modified, e.g., with a modificationdescribed herein. Modifications can include, e.g., the use ofmodifications at the 2′ position of the ribose sugar with modificationsthat are known in the art, e.g., the use of deoxyribonucleotides,2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of theribosugar of the nucleobase, and modifications in the phosphate group,e.g., phosphorothioate modifications. Overhangs need not be homologouswith the target sequence.

In some embodiments, each residue of the sense strand and antisensestrand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In certain embodiments, the N_(a) or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In some embodiments, the dsRNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′ to 3′ of the strand and the alternating motif inthe antisense strand may start with “BABABA” from 5′ to 3′ of the strandwithin the duplex region. As another example, the alternating motif inthe sense strand may start with “AABBAABB” from 5′ to 3′ of the strandand the alternating motif in the antisense strand may start with“BBAABBAA” from 5′ to 3′ of the strand within the duplex region, so thatthere is a complete or partial shift of the modification patternsbetween the sense strand and the antisense strand.

In some embodiments, the dsRNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand or antisense strandinterrupts the initial modification pattern present in the sense strandor antisense strand. This interruption of the modification pattern ofthe sense or antisense strand by introducing one or more motifs of threeidentical modifications on three consecutive nucleotides to the sense orantisense strand may enhance the gene silencing activity against thetarget gene.

In some embodiments, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)or N_(b) may be present or absent when there is a wing modificationpresent.

The iRNA may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand, antisense strand, or both strands in anyposition of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand or antisense strand; or thesense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages. In some embodiments, the antisense strandcomprises two phosphorothioate internucleotide linkages at the 5′-endand two phosphorothioate internucleotide linkages at the 3′-end, and thesense strand comprises at least two phosphorothioate internucleotidelinkages at either the 5′-end or the 3′-end.

In some embodiments, the dsRNAi agent comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, or the 5′ end of the antisense strand.

In some embodiments, the 2-nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, thedsRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mismatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of thefirst 1, 2, 3, 4, or 5 base pairs within the duplex regions from the5′-end of the antisense strand independently selected from the group of:A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other thancanonical pairings or pairings which include a universal base, topromote the dissociation of the antisense strand at the 5′-end of theduplex.

In certain embodiments, the nucleotide at the 1 position within theduplex region from the 5′-end in the antisense strand is selected fromA, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or3 base pair within the duplex region from the 5′-end of the antisensestrand is an AU base pair. For example, the first base pair within theduplex region from the 5′-end of the antisense strand is an AU basepair.

In other embodiments, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT) or the nucleotide at the 3′-end of the antisensestrand is deoxy-thymine (dT). For example, there is a short sequence ofdeoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-endof the sense, antisense strand, or both strands.

In certain embodiments, the sense strand sequence may be represented byformula (I):5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each np and n independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and XXX, YYY, andZZZ each independently represent one motif of three identicalmodifications on three consecutive nucleotides. Preferably YYY is all2′-F modified nucleotides.

In some embodiments, the N_(a) or N_(b) comprises modifications ofalternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage siteof the sense strand. For example, when the dsRNAi agent has a duplexregion of 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7,8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13) of the sensestrand, the count starting from the first nucleotide, from the 5′-end;or optionally, the count starting at the first paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′  (Ib);5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q)3′  (Ic); or5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2, or 0 modified nucleotides. Each N_(a) can independently representan oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N_(b) is 0,1, 2, 3, 4, 5, or 6 Each Na can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:5′n_(p)-N_(a)-YYY-N_(a)-n_(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):5′n_(q′)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)-N′_(a)-n_(p)′3′  (II)wherein:

k and 1 are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides; each N_(b)′ independentlyrepresents an oligonucleotide sequence comprising 0-10 modifiednucleotides;

each np′ and n′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In some embodiments, the N_(a)′ or N_(b)′ comprises modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the dsRNAi agent has a duplex region of 17-23nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10,11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the first nucleotide, from the5′-end; or optionally, the count starting at the first paired nucleotidewithin the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motifoccurs at positions 11, 12, 13.

In certain embodiments, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In certain embodiments, k is 1 and is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:5′n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p′)3′  (IIb);5′n_(q)′-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p′)3′  (Ic); or5′n_(q)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′—N_(b)′-X′X′X′-N_(a)′-n_(p′)3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5, or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may berepresented by the formula:5′n_(p′)-N_(a)′-Y′Y′Y′—N_(a)′-n_(q′)3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, CRN, UNA, cEt, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or2′-fluoro. For example, each nucleotide of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro. Each X, Y, Z, X′, Y′, and Z′, in particular, may represent a2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the dsRNAi agent may containYYY motif occurring at 9, 10, and 11 positions of the strand when theduplex region is 21 nt, the count starting from the first nucleotidefrom the 5′-end, or optionally, the count starting at the first pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In some embodiments the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe first nucleotide from the 5′-end, or optionally, the count startingat the first paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the dsRNAi agents for use in the methods of the inventionmay comprise a sense strand and an antisense strand, each strand having14 to 30 nucleotides, the iRNA duplex represented by formula (III):sense: 5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)—N_(a)-n_(q)3′antisense:3′n_(p′)-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)-N_(a)′-n_(q′)5′  (III)

wherein:

i, j, k, and 1 are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or maynot be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand formingan iRNA duplex include the formulas below:5′n_(p)-N_(a)-YYY-N_(a)-n_(q)3′3′n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q′)5′   (IIIa)5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′3′n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′n_(q′)5′   (IIIb)5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q)3′3′n_(p)′—N_(a)′-X′X′X′—N_(b)′-Y′Y′Y′—N_(a)′-n_(q′)5′   (IIIc)5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′3′n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-ng′5′   (IIId)

When the dsRNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5, or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the dsRNAi agent is represented as formula (IIIc), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. EachN_(a) independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIId), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. EachN_(a), N_(a)′ independently represents an oligonucleotide sequencecomprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a),N_(a)′, N_(b), and N_(b)′ independently comprises modifications ofalternating pattern.

Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and(IIId) may be the same or different from each other.

When the dsRNAi agent is represented by formula (III), (IIIa), (IIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the dsRNAi agent is represented by formula (IIb) or (IIId), atleast one of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNAi agent is represented as formula (IIIc) or (IIId), atleast one of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In certain embodiments, the modification on the Y nucleotide isdifferent than the modification on the Y′ nucleotide, the modificationon the Z nucleotide is different than the modification on the Z′nucleotide, or the modification on the X nucleotide is different thanthe modification on the X′ nucleotide.

In certain embodiments, when the dsRNAi agent is represented by formula(IId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications. In other embodiments, when the RNAi agent is representedby formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet otherembodiments, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GaNAc derivatives attached through a bivalent or trivalent branchedlinker (described below). In other embodiments, when the RNAi agent isrepresented by formula (IIId), the N_(a) modifications are 2′-O-methylor 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linkedto a neighboring nucleotide via phosphorothioate linkage, the sensestrand comprises at least one phosphorothioate linkage, and the sensestrand is conjugated to one or more GaNAc derivatives attached through abivalent or trivalent branched linker.

In some embodiments, when the dsRNAi agent is represented by formula(IIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications, n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via phosphorothioate linkage, the sense strandcomprises at least one phosphorothioate linkage, and the sense strand isconjugated to one or more GaNAc derivatives attached through a bivalentor trivalent branched linker.

In some embodiments, the dsRNAi agent is a multimer containing at leasttwo duplexes represented by formula (III), (IIa), (IIb), (IIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In some embodiments, the dsRNAi agent is a multimer containing three,four, five, six, or more duplexes represented by formula (III), (IIa),(IIb), (IIc), and (IIId), wherein the duplexes are connected by alinker. The linker can be cleavable or non-cleavable. Optionally, themultimer further comprises a ligand. Each of the duplexes can target thesame gene or two different genes; or each of the duplexes can targetsame gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one offormulas (III), (IIa), (IIb), (IIc), and (IIId) are linked to each otherat the 5′ end, and one or both of the 3′ ends, and are optionallyconjugated to a ligand. Each of the agents can target the same gene ortwo different genes; or each of the agents can target same gene at twodifferent target sites.

In certain embodiments, an RNAi agent of the invention may contain a lownumber of nucleotides containing a 2′-fluoro modification, e.g., 10 orfewer nucleotides with 2′-fluoro modification. For example, the RNAiagent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides with a2′-fluoro modification. In a specific embodiment, the RNAi agent of theinvention contains 10 nucleotides with a 2′-fluoro modification, e.g., 4nucleotides with a 2′-fluoro modification in the sense strand and 6nucleotides with a 2′-fluoro modification in the antisense strand. Inanother specific embodiment, the RNAi agent of the invention contains 6nucleotides with a 2′-fluoro modification, e.g., 4 nucleotides with a2′-fluoro modification in the sense strand and 2 nucleotides with a2′-fluoro modification in the antisense strand.

In other embodiments, an RNAi agent of the invention may contain anultra low number of nucleotides containing a 2′-fluoro modification,e.g., 2 or fewer nucleotides containing a 2′-fluoro modification. Forexample, the RNAi agent may contain 2, 1 of 0 nucleotides with a2′-fluoro modification. In a specific embodiment, the RNAi agent maycontain 2 nucleotides with a 2′-fluoro modification, e.g., 0 nucleotideswith a 2-fluoro modification in the sense strand and 2 nucleotides witha 2′-fluoro modification in the antisense strand.

Various publications describe multimeric iRNAs that can be used in themethods of the invention. Such publications include WO2007/091269, U.S.Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the iRNA that contains conjugationsof one or more carbohydrate moieties to an iRNA can optimize one or moreproperties of the iRNA. In many cases, the carbohydrate moiety will beattached to a modified subunit of the iRNA. For example, the ribosesugar of one or more ribonucleotide subunits of a iRNA can be replacedwith another moiety, e.g., a non-carbohydrate (preferably cyclic)carrier to which is attached a carbohydrate ligand. A ribonucleotidesubunit in which the ribose sugar of the subunit has been so replaced isreferred to herein as a ribose replacement modification subunit (RRMS).A cyclic carrier may be a carbocyclic ring system, i.e., all ring atomsare carbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The iRNA may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, anddecalin; preferably, the acyclic group is a serinol backbone ordiethanolamine backbone.

In another embodiment of the invention, an iRNA agent comprises a sensestrand and an antisense strand, each strand having 14 to 40 nucleotides.The RNAi agent may be represented by formula (L):

In formula (L), B1, B2, B3, B1′, B2′, B3′, and B4′ each areindependently a nucleotide containing a modification selected from thegroup consisting of 2′-O-alkyl, 2′-substituted alkoxy, 2′-substitutedalkyl, 2′-halo, ENA, and BNA/LNA. In one embodiment, B1, B2, B3, B1′,B2′, B3′, and B4′ each contain 2′-OMe modifications. In one embodiment,B1, B2, B3, B1′, B2′, B3′, and B4′ each contain 2′-OMe or 2′-Fmodifications. In one embodiment, at least one of B1, B2, B3, B1′, B2′,B3′, and B4′ contain 2′-O—N-methylacetamido (2′-O-NMA) modification.

C1 is a thermally destabilizing nucleotide placed at a site opposite tothe seed region of the antisense strand (i.e., at positions 2-8 of the5′-end of the antisense strand). For example, C1 is at a position of thesense strand that pairs with a nucleotide at positions 2-8 of the 5′-endof the antisense strand. In one example, C1 is at position 15 from the5′-end of the sense strand. C1 nucleotide bears the thermallydestabilizing modification which can include abasic modification;mismatch with the opposing nucleotide in the duplex; and sugarmodification such as 2′-deoxy modification or acyclic nucleotide e.g.,unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA). In oneembodiment, C1 has thermally destabilizing modification selected fromthe group consisting of: i) mismatch with the opposing nucleotide in theantisense strand; ii) abasic modification selected from the groupconsisting of:

and iii) sugar modification selected from the group consisting of:

wherein B is a modified or unmodified nucleobase, R¹ and R²independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In one embodiment, thethermally destabilizing modification in C1 is a mismatch selected fromthe group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T,U:U, T:T, and U:T; and optionally, at least one nucleobase in themismatch pair is a 2′-deoxy nucleobase. In one example, the thermallydestabilizing modification in C1 is GNA or

T1, T1′, T2′, and T3′ each independently represent a nucleotidecomprising a modification providing the nucleotide a steric bulk that isless or equal to the steric bulk of a 2′-OMe modification. A steric bulkrefers to the sum of steric effects of a modification. Methods fordetermining steric effects of a modification of a nucleotide are knownto one skilled in the art. The modification can be at the 2′ position ofa ribose sugar of the nucleotide, or a modification to a non-ribosenucleotide, acyclic nucleotide, or the backbone of the nucleotide thatis similar or equivalent to the 2′ position of the ribose sugar, andprovides the nucleotide a steric bulk that is less than or equal to thesteric bulk of a 2′-OMe modification. For example, T1, T1′, T2′, and T3′are each independently selected from DNA, RNA, LNA, 2′-F, and2′-F-5′-methyl. In one embodiment, T1 is DNA. In one embodiment, T1′ isDNA, RNA or LNA. In one embodiment, T2′ is DNA or RNA. In oneembodiment, T3′ is DNA or RNA.n¹, n³, and q¹ are independently 4 to 15 nucleotides in length.n⁵, q³, and q⁷ are independently 1-6 nucleotide(s) in length.n⁴, q², and q⁶ are independently 1-3 nucleotide(s) in length;alternatively, n⁴ is 0.q⁵ is independently 0-10 nucleotide(s) in length.n² and q⁴ are independently 0-3 nucleotide(s) in length.

Alternatively, n⁴ is 0-3 nucleotide(s) in length.

In one embodiment, n⁴ can be 0. In one example, n⁴ is 0, and q² and q⁶are 1. In another example, n⁴ is 0, and q² and q⁶ are 1, with twophosphorothioate internucleotide linkage modifications within position1-5 of the sense strand (counting from the 5′-end of the sense strand),and two phosphorothioate internucleotide linkage modifications atpositions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end of the antisense strand).

In one embodiment, n⁴, q², and q⁶ are each 1.

In one embodiment, n², n⁴ q² 4, and q⁶ are each 1.

In one embodiment, C1 is at position 14-17 of the 5′-end of the sensestrand, when the sense strand is 19-22 nucleotides in length, and n⁴is 1. In one embodiment, C1 is at position 15 of the 5′-end of the sensestrand

In one embodiment, T3′ starts at position 2 from the 5′ end of theantisense strand. In one example, T3′ is at position 2 from the 5′ endof the antisense strand and q⁶ is equal to 1.

In one embodiment, T1′ starts at position 14 from the 5′ end of theantisense strand. In one example, T1′ is at position 14 from the 5′ endof the antisense strand and q² is equal to 1.

In an exemplary embodiment, T3′ starts from position 2 from the 5′ endof the antisense strand and T1′ starts from position 14 from the 5′ endof the antisense strand. In one example, T3′ starts from position 2 fromthe 5′ end of the antisense strand and q⁶ is equal to 1 and T1′ startsfrom position 14 from the 5′ end of the antisense strand and q² is equalto 1.

In one embodiment, T1′ and T3′ are separated by 11 nucleotides in length(i.e. not counting the T1′ and T3′ nucleotides).

In one embodiment, T1′ is at position 14 from the 5′ end of theantisense strand. In one example, T1′ is at position 14 from the 5′ endof the antisense strand and q² is equal to 1, and the modification atthe 2′ position or positions in a non-ribose, acyclic or backbone thatprovide less steric bulk than a 2′-OMe ribose.

In one embodiment, T3′ is at position 2 from the 5′ end of the antisensestrand. In one example, T3′ is at position 2 from the 5′ end of theantisense strand and q⁶ is equal to 1, and the modification at the 2′position or positions in a non-ribose, acyclic or backbone that provideless than or equal to steric bulk than a 2′-OMe ribose.

In one embodiment, T1 is at the cleavage site of the sense strand. Inone example, T1 is at position 11 from the 5′ end of the sense strand,when the sense strand is 19-22 nucleotides in length, and n² is 1. In anexemplary embodiment, T1 is at the cleavage site of the sense strand atposition 11 from the 5′ end of the sense strand, when the sense strandis 19-22 nucleotides in length, and n² is 1, In one embodiment, T2′starts at position 6 from the 5′ end of the antisense strand. In oneexample, T2′ is at positions 6-10 from the 5′ end of the antisensestrand, and q⁴ is 1.

In an exemplary embodiment, T1 is at the cleavage site of the sensestrand, for instance, at position 11 from the 5′ end of the sensestrand, when the sense strand is 19-22 nucleotides in length, and n² is1; T1′ is at position 14 from the 5′ end of the antisense strand, and q²is equal to 1, and the modification to T1′ is at the 2′ position of aribose sugar or at positions in a non-ribose, acyclic or backbone thatprovide less steric bulk than a 2′-OMe ribose; T2′ is at positions 6-10from the 5′ end of the antisense strand, and q⁴ is 1; and T3′ is atposition 2 from the 5′ end of the antisense strand, and q⁶ is equal to1, and the modification to T3′ is at the 2′ position or at positions ina non-ribose, acyclic or backbone that provide less than or equal tosteric bulk than a 2′-OMe ribose. In one embodiment, T2′ starts atposition 8 from the 5′ end of the antisense strand. In one example, T2′starts at position 8 from the 5′ end of the antisense strand, and q⁴ is2.

In one embodiment, T2′ starts at position 9 from the 5′ end of theantisense strand. In one example, T2′ is at position 9 from the 5′ endof the antisense strand, and q⁴ is 1.

In one embodiment, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1,B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; withtwo phosphorothioate internucleotide linkage modifications withinpositions 1-5 of the sense strand (counting from the 5′-end of the sensestrand), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end of the antisense strand).

In one embodiment, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q′ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q′ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q′ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q′ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q′ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q′ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 5, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; optionally with at least 2 additional TT atthe 3′-end of the antisense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 5, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; optionally with at least 2 additional TT atthe 3′-end of the antisense strand; with two phosphorothioateinternucleotide linkage modifications within positions 1-5 of the sensestrand (counting from the 5′-end of the sense strand), and twophosphorothioate internucleotide linkage modifications at positions 1and 2 and two phosphorothioate internucleotide linkage modificationswithin positions 18-23 of the antisense strand (counting from the 5′-endof the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within positions 1-5 of the sense strand (counting fromthe 5′-end), and two phosphorothioate internucleotide linkagemodifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n′ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q′ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within positions 1-5 of the sense strand (counting fromthe 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

The RNAi agent can comprise a phosphorus-containing group at the 5′-endof the sense strand or antisense strand. The 5′-endphosphorus-containing group can be 5′-end phosphate (5′-P), 5′-endphosphorothioate (5′-PS), 5′-end phosphorodithioate (5′-PS₂), 5′-endvinylphosphonate (5′-VP), 5′-end methylphosphonate (MePhos), or5′-deoxy-5′-C-malonyl

When the 5′-end phosphorus-containing group is 5′-end vinylphosphonate(5′-VP), the 5′-VP can be either 5′-E-VP isomer (i.e.,trans-vinylphosphate.

5′-Z-VP isomer (i.e., cis-vinylphosphate,

or mixtures thereof.

In one embodiment, the RNAi agent comprises a phosphorus-containinggroup at the 5′-end of the sense strand. In one embodiment, the RNAiagent comprises a phosphorus-containing group at the 5′-end of theantisense strand.

In one embodiment, the RNAi agent comprises a 5′-P. In one embodiment,the RNAi agent comprises a 5′-P in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS. In one embodiment,the RNAi agent comprises a 5′-PS in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-VP. In one embodiment,the RNAi agent comprises a 5′-VP in the antisense strand. In oneembodiment, the RNAi agent comprises a 5′-E-VP in the antisense strand.In one embodiment, the RNAi agent comprises a 5′-Z-VP in the antisensestrand.

In one embodiment, the RNAi agent comprises a 5′-PS₂. In one embodiment,the RNAi agent comprises a 5′-PS₂ in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS₂. In one embodiment,the RNAi agent comprises a 5′-deoxy-5′-C-malonyl in the antisensestrand. In one embodiment, B1 is 2′-OMe or 2′-F, is 8, T1 is 2′F, n² is3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMeor 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4,53 T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VPmay be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The RNAi agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-VP. The 5′-VP maybe 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The dsRNAi RNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, orcombination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-P and a targeting ligand. Inone embodiment, the 5′-P is at the 5′-end of the antisense strand, andthe targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS and a targeting ligand.In one embodiment, the 5′-PS is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP,5′-Z-VP, or combination thereof), and a targeting ligand.

In one embodiment, the 5′-VP is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand.In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and atargeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the5′-end of the antisense strand, and the targeting ligand is at the3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-P and a targetingligand. In one embodiment, the 5′-P is at the 5′-end of the antisensestrand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-PS and a targetingligand. In one embodiment, the 5′-PS is at the 5′-end of the antisensestrand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-VP (e.g., a5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In oneembodiment, the 5′-VP is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-PS₂ and a targetingligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisensestrand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyland a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl isat the 5′-end of the antisense strand, and the targeting ligand is atthe 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-P and a targeting ligand. Inone embodiment, the 5′-P is at the 5′-end of the antisense strand, andthe targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS and a targeting ligand.In one embodiment, the 5′-PS is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP,5′-Z-VP, or combination thereof) and a targeting ligand. In oneembodiment, the 5′-VP is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand.In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and atargeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the5′-end of the antisense strand, and the targeting ligand is at the3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-P and a targeting ligand. In oneembodiment, the 5′-P is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-PS and a targeting ligand. In oneembodiment, the 5′-PS is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, orcombination thereof) and a targeting ligand. In one embodiment, the5′-VP is at the 5′-end of the antisense strand, and the targeting ligandis at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-PS₂ and a targeting ligand. In oneembodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targetingligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end ofthe antisense strand, and the targeting ligand is at the 3′-end of thesense strand.

In a particular embodiment, an RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker; and        -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,            13, 17, 19, and 21, and 2′-OMe modifications at positions 2,            4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5′            end); and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 3, 5, 9, 11 to 13,            15, 17, 19, 21, and 23, and 2′F modifications at positions            2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the            5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 21 and 22, and between nucleotide            positions 22 and 23 (counting from the 5′ end);    -   wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.    -   In another particular embodiment, an RNAi agent of the present        invention comprises:    -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,            13, 15, 17, 19, and 21, and 2′-OMe modifications at            positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from            the 5′ end); and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end); and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to            13, 15, 17, 19, and 21 to 23, and 2′F modifications at            positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from            the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.    -   In another particular embodiment, a RNAi agent of the present        invention comprises:    -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, and            12 to 21, 2′-F modifications at positions 7, and 9, and a            deoxy-nucleotide (e.g. dT) at position 11 (counting from the            5′ end); and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end); and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 3, 7, 9, 11, 13,            15, 17, and 19 to 23, and 2′-F modifications at positions 2,            4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5′            end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.    -   In another particular embodiment, a RNAi agent of the present        invention comprises:    -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, 12,            14, and 16 to 21, and 2′-F modifications at positions 7, 9,            11, 13, and 15; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end); and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 5, 7, 9, 11, 13,            15, 17, 19, and 21 to 23, and 2′-F modifications at            positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting            from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.    -   In another particular embodiment, a RNAi agent of the present        invention comprises:    -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 9, and 12 to            21, and 2′-F modifications at positions 10, and 11; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end); and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to            13, 15, 17, 19, and 21 to 23, and 2′-F modifications at            positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from            the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.    -   In another particular embodiment, a RNAi agent of the present        invention comprises:    -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,            and 13, and 2′-OMe modifications at positions 2, 4, 6, 8,            12, and 14 to 21; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end); and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 3, 5 to 7, 9, 11            to 13, 15, 17 to 19, and 21 to 23, and 2′-F modifications at            positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5′            end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.    -   In another particular embodiment, a RNAi agent of the present        invention comprises:    -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1, 2, 4, 6, 8, 12,            14, 15, 17, and 19 to 21, and 2′-F modifications at            positions 3, 5, 7, 9 to 11, 13, 16, and 18; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end); and    -   (b) an antisense strand having:        -   (i) a length of 25 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 4, 6, 7, 9, 11 to            13, 15, 17, and 19 to 23, 2′-F modifications at positions 2,            3, 5, 8, 10, 14, 16, and 18, and desoxy-nucleotides (e.g.            dT) at positions 24 and 25 (counting from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a four nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.    -   In another particular embodiment, a RNAi agent of the present        invention comprises:    -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to            21, and 2′-F modifications at positions 7, and 9 to 11; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end); and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 8, 10            to 13, 15, and 17 to 23, and 2′-F modifications at positions            2, 6, 9, 14, and 16 (counting from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.    -   In another particular embodiment, a RNAi agent of the present        invention comprises:    -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to            21, and 2′-F modifications at positions 7, and 9 to 11; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end); and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to            13, 15, and 17 to 23, and 2′-F modifications at positions 2,            6, 8, 9, 14, and 16 (counting from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 19 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 4, 6, and 10 to            19, and 2′-F modifications at positions 5, and 7 to 9; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end); and    -   (b) an antisense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to            13, 15, and 17 to 21, and 2′-F modifications at positions 2,            6, 8, 9, 14, and 16 (counting from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 19 and 20, and between            nucleotide positions 20 and 21 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In certain embodiments, the iRNA for use in the methods of the inventionis an agent selected from agents listed in Table 3, Table 5, or Table 6.These agents may further comprise a ligand.

III. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the iRNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution, or cellularuptake of the iRNA e.g., into a cell. Such moieties include but are notlimited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556). In otherembodiments, the ligand is cholic acid (Manoharan et al., Biorg. Med.Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol(Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660:306-309; Manoharanet al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990,259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54),aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting, orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands do nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, orhyaluronic acid); or a lipid. The ligand can also be a recombinant orsynthetic molecule, such as a synthetic polymer, e.g., a syntheticpolyamino acid. Examples of polyamino acids include polyamino acid is apolylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic. In certain embodiments, the ligand is amultivalent galactose, e.g., an N-acetyl-galactosamine.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, or intermediate filaments. The drug can be, for example,taxol, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins, etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.,oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases,comprising multiple of phosphorothioate linkages in the backbone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands). In addition, aptamers that bind serum components (e.g. serumproteins) are also suitable for use as PK modulating ligands in theembodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the useof an oligonucleotide that bears a pendant reactive functionality, suchas that derived from the attachment of a linking molecule onto theoligonucleotide (described below). This reactive oligonucleotide may bereacted directly with commercially-available ligands, ligands that aresynthesized bearing any of a variety of protecting groups, or ligandsthat have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems® (Foster City,Calif.). Any other methods for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated iRNAs and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In certain embodiments, the ligand or conjugate is a lipid orlipid-based molecule. Such a lipid or lipid-based molecule preferablybinds a serum protein, e.g., human serum albumin (HSA). An HSA bindingligand allows for distribution of the conjugate to a target tissue,e.g., a non-kidney target tissue of the body. For example, the targettissue can be the liver, including parenchymal cells of the liver. Othermolecules that can bind HSA can also be used as ligands. For example,naproxen or aspirin can be used. A lipid or lipid-based ligand can (a)increase resistance to degradation of the conjugate, (b) increasetargeting or transport into a target cell or cell membrane, or (c) canbe used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In certain embodiments, the lipid based ligand binds HSA. Preferably, itbinds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In other embodiments, the lipid based ligand binds HSA weakly or not atall, such that the conjugate will be preferably distributed to thekidney. Other moieties that target to kidney cells can also be used inplace of, or in addition to, the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 15). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:16) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:17) and theDrosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:18) havebeen found to be capable of functioning as delivery peptides. A peptideor peptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA further comprises a carbohydrate. The carbohydrate conjugated iRNAis advantageous for the in vivo delivery of nucleic acids, as well ascompositions suitable for in vivo therapeutic use, as described herein.As used herein, “carbohydrate” refers to a compound which is either acarbohydrate per se made up of one or more monosaccharide units havingat least 6 carbon atoms (which can be linear, branched or cyclic) withan oxygen, nitrogen or sulfur atom bonded to each carbon atom; or acompound having as a part thereof a carbohydrate moiety made up of oneor more monosaccharide units each having at least six carbon atoms(which can be linear, branched or cyclic), with an oxygen, nitrogen orsulfur atom bonded to each carbon atom. Representative carbohydratesinclude the sugars (mono-, di-, tri-, and oligosaccharides containingfrom about 4, 5, 6, 7, 8, or 9 monosaccharide units), andpolysaccharides such as starches, glycogen, cellulose and polysaccharidegums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7,or C8) sugars; di- and trisaccharides include sugars having two or threemonosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate for use in thecompositions and methods of the invention is a monosaccharide.

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is selected from the group consisting of:

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is a monosaccharide. In oneembodiment, the monosaccharide is an N-acetylgalactosamine, such as

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the invention, the GalNAc or GalNAc derivativeis attached to an iRNA agent of the invention via a monovalent linker.In some embodiments, the GaNAc or GaNAc derivative is attached to aniRNA agent of the invention via a bivalent linker. In yet otherembodiments of the invention, the GalNAc or GalNAc derivative isattached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the inventioncomprise one GaNAc or GalNAc derivative attached to the iRNA agent,e.g., the 5′ end of the sense strand of a dsRNA agent, or the 5′ end ofone or both sense strands of a dual targeting RNAi agent as describedherein.

In another embodiment, the double stranded RNAi agents of the inventioncomprise a plurality (e.g., 2, 3, 4, 5, or 6) GaNAc or GaNAcderivatives, each independently attached to a plurality of nucleotidesof the double stranded RNAi agent through a plurality of monovalentlinkers.

In some embodiments, for example, when the two strands of an iRNA agentof the invention are part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates and linkers suitable for use in thepresent invention include those described in PCT Publication Nos. WO2014/179620 and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, or substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, orsubstituted aliphatic. In one embodiment, the linker is about 1-24atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16,7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times, or more, or at least 100 times faster in a target cell or under afirst reference condition (which can, e.g., be selected to mimic orrepresent intracellular conditions) than in the blood of a subject, orunder a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential, or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100times faster in the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood or serum (or under invitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In certain embodiments, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In other embodiments, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—,—S—P(O)(H)—S—, and —O—P(S)(H)—S—. A preferred embodiment is—O—P(O)(OH)—O—. These candidates can be evaluated using methodsanalogous to those described above.

iii. Acid Cleavable Linking Groups

In other embodiments, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or byagents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In other embodiments, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include, but are not limited to,esters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet other embodiments, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include but are not limited to

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XLV)-(XLVI):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl; L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A),L^(5B) and L^(5C) represent the ligand; i.e. each independently for eachoccurrence a monosaccharide (such as GaNAc), disaccharide,trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; andR^(a) is H or amino acid side chain. Trivalent conjugating GalNAcderivatives are particularly useful for use with RNAi agents forinhibiting the expression of a target gene, such as those of formula(XLIX):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GaNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. Patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; and 8,106,022, the entire contents of each ofwhich are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAi agents, that containtwo or more chemically distinct regions, each made up of at least onemonomer unit, i.e., a nucleotide in the case of a dsRNA compound. TheseiRNAs typically contain at least one region wherein the RNA is modifiedso as to confer upon the iRNA increased resistance to nucleasedegradation, increased cellular uptake, or increased binding affinityfor the target nucleic acid. An additional region of the iRNA can serveas a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of iRNA inhibition of gene expression.Consequently, comparable results can often be obtained with shorteriRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), athioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof RNAs bearing an aminolinker at one or more positions of the sequence.The amino group is then reacted with the molecule being conjugated usingappropriate coupling or activating reagents. The conjugation reactioncan be performed either with the RNA still bound to the solid support orfollowing cleavage of the RNA, in solution phase. Purification of theRNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject susceptible to or diagnosed with an AGT associateddisorder, e.g., hypertension) can be achieved in a number of differentways. For example, delivery may be performed by contacting a cell withan iRNA of the invention either in vitro or in vivo. In vivo deliverymay also be performed directly by administering a composition comprisingan iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo deliverymay be performed indirectly by administering one or more vectors thatencode and direct the expression of the iRNA. These alternatives arediscussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. RNAinterference has also shown success with local delivery to the CNS bydirect injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMCNeurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528;Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J Neurophysiol. 93:594-602).Modification of the RNA or the pharmaceutical carrier can also permittargeting of the iRNA to the target tissue and avoid undesirableoff-target effects. iRNA molecules can be modified by chemicalconjugation to lipophilic groups such as cholesterol to enhance cellularuptake and prevent degradation. For example, an iRNA directed againstApoB conjugated to a lipophilic cholesterol moiety was injectedsystemically into mice and resulted in knockdown of apoB mRNA in boththe liver andjejunum (Soutschek, J., et al (2004) Nature 432:173-178).

In an alternative embodiment, the iRNA can be delivered using drugdelivery systems such as a nanoparticle, a dendrimer, a polymer,liposomes, or a cationic delivery system. Positively charged cationicdelivery systems facilitate binding of an iRNA molecule (negativelycharged) and also enhance interactions at the negatively charged cellmembrane to permit efficient uptake of an iRNA by the cell. Cationiclipids, dendrimers, or polymers can either be bound to an iRNA, orinduced to form a vesicle or micelle (see e.g., Kim S H, et al (2008)Journal of Controlled Release 129(2):107-116) that encases an iRNA. Theformation of vesicles or micelles further prevents degradation of theiRNA when administered systemically. Methods for making andadministering cationic-iRNA complexes are well within the abilities ofone skilled in the art (see e.g., Sorensen, D R, et al (2003) J. Mol.Biol 327:761-766; Verma, U N, et al (2003) Clin. Cancer Res.9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, whichare incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N, et al (2003), supra), “solid nucleic acid lipid particles”(Zimmermann, T S, et al (2006) Nature 441:111-114), cardiolipin (Chien,P Y, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) IntJ Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E, et al (2008)Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed.Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol.Pharm. 3:472-487), and polyamidoamines (Tomalia, D A, et al (2007)Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res.16:1799-1804). In some embodiments, an iRNA forms a complex withcyclodextrin for systemic administration. Methods for administration andpharmaceutical compositions of iRNAs and cyclodextrins can be found inU.S. Pat. No. 7,427,605, which is herein incorporated by reference inits entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the AGT gene can be expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A, et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). Expression can be transient (on theorder of hours to weeks) or sustained (weeks to months or longer),depending upon the specific construct used and the target tissue or celltype. These transgenes can be introduced as a linear construct, acircular plasmid, or a viral vector, which can be an integrating ornon-integrating vector. The transgene can also be constructed to permitit to be inherited as an extrachromosomal plasmid (Gassmann, et al.,Proc. Natl. Acad. Sci. USA (1995) 92:1292).

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful forpreventing or treating an AGT associated disorder, e.g., hypertension.Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by subcutaneous (SC),intramuscular (IM), or intravenous (IV) delivery. The pharmaceuticalcompositions of the invention may be administered in dosages sufficientto inhibit expression of an AGT gene.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of an AGT gene. In general, asuitable dose of an iRNA of the invention will be in the range of about0.001 to about 200.0 milligrams per kilogram body weight of therecipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. Typically, a suitable dose of an iRNA ofthe invention will be in the range of about 0.1 mg/kg to about 5.0mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg. A repeat-doseregimen may include administration of a therapeutic amount of iRNA on aregular basis, such as every month, once every 3-6 months, or once ayear. In certain embodiments, the iRNA is administered about once permonth to about once per six months.

After an initial treatment regimen, the treatments can be administeredon a less frequent basis. Duration of treatment can be determined basedon the severity of disease.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that doses are administered at not more than1, 2, 3, or 4 month intervals. In some embodiments of the invention, asingle dose of the pharmaceutical compositions of the invention isadministered about once per month. In other embodiments of theinvention, a single dose of the pharmaceutical compositions of theinvention is administered quarterly (i.e., about every three months). Inother embodiments of the invention, a single dose of the pharmaceuticalcompositions of the invention is administered twice per year (i.e.,about once every six months).

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to mutations present in the subject, previoustreatments, the general health or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a prophylactically ortherapeutically effective amount, as appropriate, of a composition caninclude a single treatment or a series of treatments.

The iRNA can be delivered in a manner to target a particular tissue(e.g., hepatocytes).

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids, and self-emulsifying semisolids. Formulationsinclude those that target the liver.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers.

A. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution either in the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Other means of stabilizing emulsions entail the use ofemulsifiers that can be incorporated into either phase of the emulsion.Emulsifiers can broadly be classified into four categories: syntheticsurfactants, naturally occurring emulsifiers, absorption bases, andfinely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Formsand Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C.,2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic, and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives, andantioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

The application of emulsion formulations via dermatological, oral, andparenteral routes, and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil, and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215).

iii. Microparticles

An iRNA of the invention may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying, but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers and their use in manufacture of pharmaceuticalcompositions and delivery of pharmaceutical agents are well known in theart.

v. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agent,or any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Such agent are well known in the art.

vi. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavorings,or aromatic substances, and the like which do not deleteriously interactwith the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol, or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA and (b) one or more agents whichfunction by a non-iRNA mechanism and which are useful in treating an AGTassociated disorder, e.g., hypertension.

Toxicity and prophylactic efficacy of such compounds can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose prophylactically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50, preferably anED80 or ED90, with little or no toxicity. The dosage can vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound used in the methods featuredin the invention, the prophylactically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range of thecompound or, when appropriate, of the polypeptide product of a targetsequence (e.g., achieving a decreased concentration of the polypeptide)that includes the IC50 (i.e., the concentration of the test compoundwhich achieves a half-maximal inhibition of symptoms) or higher levelsof inhibition as determined in cell culture. Such information can beused to more accurately determine useful doses in humans. Levels inplasma can be measured, for example, by high performance liquidchromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents used for the prevention or treatment of an AGT associateddisorder, e.g., hypertension. In any event, the administering physiciancan adjust the amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

VI. Methods for Inhibiting AGT Expression

The present invention also provides methods of inhibiting expression ofan AGT gene in a cell. The methods include contacting a cell with anRNAi agent, e.g., double stranded RNA agent, in an amount effective toinhibit expression of AGT in the cell, thereby inhibiting expression ofAGT in the cell.

Contacting of a cell with an iRNA, e.g., a double stranded RNA agent,may be done in vitro or in vivo. Contacting a cell in vivo with the iRNAincludes contacting a cell or group of cells within a subject, e.g., ahuman subject, with the iRNA. Combinations of in vitro and in vivomethods of contacting a cell are also possible. Contacting a cell may bedirect or indirect, as discussed above. Furthermore, contacting a cellmay be accomplished via a targeting ligand, including any liganddescribed herein or known in the art. In preferred embodiments, thetargeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, orany other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating”, “suppressing”, and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of an AGT” is intended to refer toinhibition of expression of any AGT gene (such as, e.g., a mouse AGTgene, a rat AGT gene, a monkey AGT gene, or a human AGT gene) as well asvariants or mutants of an AGTgene. Thus, the AGT gene may be a wild-typeAGT gene, a mutant AGT gene, or a transgenic AGT gene in the context ofa genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of an AGT gene” includes any level of inhibitionof an AGT gene, e.g., at least partial suppression of the expression ofan AGT gene. The expression of the AGT gene may be assessed based on thelevel, or the change in the level, of any variable associated with AGTgene expression, e.g., AGT mRNA level or AGT protein level. This levelmay be assessed in an individual cell or in a group of cells, including,for example, a sample derived from a subject. It is understood that AGTis expressed predominantly in the liver, but also in the brain, gallbladder, heart, and kidney, and is present in circulation.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with AGT expressioncompared with a control level. The control level may be any type ofcontrol level that is utilized in the art, e.g., a pre-dose baselinelevel, or a level determined from a similar subject, cell, or samplethat is untreated or treated with a control (such as, e.g., buffer onlycontrol or inactive agent control).

In some embodiments of the methods of the invention, expression of anAGT gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95%, or to below the level of detection of the assay. Inpreferred embodiments, expression of an AGT gene is inhibited by atleast 70%. It is further understood that inhibition of AGT expression incertain tissues, e.g., in liver, without a significant inhibition ofexpression in other tissues, e.g., brain, may be desirable. In preferredembodiments, expression level is determined using the assay methodprovided in Example 2 with a 10 nM siRNA concentration in theappropriate species matched cell line.

In certain embodiments, inhibition of expression in vivo is determinedby knockdown of the human gene in a rodent expressing the human gene,e.g., an AAV-infected mouse expressing the human target gene (i.e.,AGT), e.g., when administered a single dose at 3 mg/kg at the nadir ofRNA expression. Knockdown of expression of an endogenous gene in a modelanimal system can also be determined, e.g., after administration of asingle dose at 3 mg/kg at the nadir of RNA expression. Such systems areuseful when the nucleic acid sequence of the human gene and the modelanimal gene are sufficiently close such that the human iRNA provideseffective knockdown of the model animal gene. RNA expression in liver isdetermined using the PCR methods provided in Example 2.

Inhibition of the expression of an AGT gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which an AGT gene is transcribed and which has or havebeen treated (e.g., by contacting the cell or cells with an iRNA of theinvention, or by administering an iRNA of the invention to a subject inwhich the cells are or were present) such that the expression of an AGTgene is inhibited, as compared to a second cell or group of cellssubstantially identical to the first cell or group of cells but whichhas not or have not been so treated (control cell(s) not treated with aniRNA or not treated with an iRNA targeted to the gene of interest). Inpreferred embodiments, the inhibition is assessed by the method providedin Example 2 using a 10 nM siRNA concentration in the species matchedcell line and expressing the level of mRNA in treated cells as apercentage of the level of mRNA in control cells, using the followingformula:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of an AGT gene may beassessed in terms of a reduction of a parameter that is functionallylinked to AGT gene expression, e.g., AGT protein level in blood or serumfrom a subject. AGT gene silencing may be determined in any cellexpressing AGT, either endogenous or heterologous from an expressionconstruct, and by any assay known in the art.

Inhibition of the expression of an AGT protein may be manifested by areduction in the level of the AGT protein that is expressed by a cell orgroup of cells or in a subject sample (e.g., the level of protein in ablood sample derived from a subject). As explained above, for theassessment of mRNA suppression, the inhibition of protein expressionlevels in a treated cell or group of cells may similarly be expressed asa percentage of the level of protein in a control cell or group ofcells, or the change in the level of protein in a subject sample, e.g.,blood or serum derived therefrom.

A control cell, a group of cells, or subject sample that may be used toassess the inhibition of the expression of an AGT gene includes a cell,group of cells, or subject sample that has not yet been contacted withan RNAi agent of the invention. For example, the control cell, group ofcells, or subject sample may be derived from an individual subject(e.g., a human or animal subject) prior to treatment of the subject withan RNAi agent or an appropriately matched population control.

The level of AGT mRNA that is expressed by a cell or group of cells maybe determined using any method known in the art for assessing mRNAexpression. In one embodiment, the level of expression of AGT in asample is determined by detecting a transcribed polynucleotide, orportion thereof, e.g., mRNA of the AGT gene. RNA may be extracted fromcells using RNA extraction techniques including, for example, using acidphenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis),RNeasy™ RNA preparation kits (Qiagen®) or PAXgene™ (PreAnalytix™,Switzerland). Typical assay formats utilizing ribonucleic acidhybridization include nuclear run-on assays, RT-PCR, RNase protectionassays, northern blotting, in situ hybridization, and microarrayanalysis.

In some embodiments, the level of expression of AGT is determined usinga nucleic acid probe. The term “probe”, as used herein, refers to anymolecule that is capable of selectively binding to a specific AGT.Probes can be synthesized by one of skill in the art, or derived fromappropriate biological preparations. Probes may be specifically designedto be labeled. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to AGTmRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix® gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of AGT mRNA.

An alternative method for determining the level of expression of AGT ina sample involves the process of nucleic acid amplification or reversetranscriptase (to prepare cDNA) of for example mRNA in the sample, e.g.,by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S.Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Nat.Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh et al. (1989) Proc. Nat.Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988)Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S.Pat. No. 5,854,033) or any other nucleic acid amplification method,followed by the detection of the amplified molecules using techniqueswell known to those of skill in the art. These detection schemes areespecially useful for the detection of nucleic acid molecules if suchmolecules are present in very low numbers. In particular aspects of theinvention, the level of expression of AGT is determined by quantitativefluorogenic RT-PCR (i.e., the TaqMan™ System). In preferred embodiments,expression level is determined by the method provided in Example 2 usinga 10 nM siRNA concentration in the species matched cell line.

The expression levels of AGT mRNA may be monitored using a membrane blot(such as used in hybridization analysis such as northern, Southern, dot,and the like), or microwells, sample tubes, gels, beads or fibers (orany solid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of AGT expressionlevel may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.In preferred embodiments, expression level is determined by the methodprovided in Example 2 using a 10 nM siRNA concentration in the speciesmatched cell line.

The level of AGT protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, western blotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention areassessed by a decrease in AGT mRNA or protein level (e.g., in a liverbiopsy).

In some embodiments of the methods of the invention, the iRNA isadministered to a subject such that the iRNA is delivered to a specificsite within the subject. The inhibition of expression of AGT may beassessed using measurements of the level or change in the level of AGTmRNA or agt protein in a sample derived from fluid or tissue from thespecific site within the subject (e.g., liver or blood).

As used herein, the terms detecting or determining a level of an analyteare understood to mean performing the steps to determine if a material,e.g., protein, RNA, is present. As used herein, methods of detecting ordetermining include detection or determination of an analyte level thatis below the level of detection for the method used.

VII. Prohylactic and Treatment Methods of the Invention

The present invention also provides methods of using an iRNA of theinvention or a composition containing an iRNA of the invention toinhibit expression of AGT, thereby preventing or treating a an AGTassociated disorder, e.g., high blood pressure, e.g., hypertension.

In the methods of the invention the cell may be contacted with the siRNAin vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an AGT gene, e.g., a liver cell, a brain cell, agall bladder cell, a heart cell, or a kidney cell, but preferably aliver cell. A cell suitable for use in the methods of the invention maybe a mammalian cell, e.g., a primate cell (such as a human cell,including human cell in a chimeric non-human animal, or a non-humanprimate cell, e.g., a monkey cell or a chimpanzee cell), or anon-primate cell. In certain embodiments, the cell is a human cell,e.g., a human liver cell. In the methods of the invention, AGTexpression is inhibited in the cell by at least 50, 55, 60, 65, 70, 75,80, 85, 90, or 95, or to a level below the level of detection of theassay.

The in vivo methods of the invention may include administering to asubject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the AGT gene of the mammal to which the RNAi agent is tobe administered. The composition can be administered by any means knownin the art including, but not limited to oral, intraperitoneal, orparenteral routes, including intracranial (e.g., intraventricular,intraparenchymal, and intrathecal), intravenous, intramuscular,subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by intravenous infusionor injection. In certain embodiments, the compositions are administeredby subcutaneous injection. In certain embodiments, the compositions areadministered by intramuscular injection.

In one aspect, the present invention also provides methods forinhibiting the expression of an AGTgene in a mammal. The methods includeadministering to the mammal a composition comprising a dsRNA thattargets an AGT gene in a cell of the mammal and maintaining the mammalfor a time sufficient to obtain degradation of the mRNA transcript ofthe AGT gene, thereby inhibiting expression of the AGT gene in the cell.Reduction in gene expression can be assessed by any methods known in theart and by methods, e.g. qRT-PCR, described herein, e.g., in Example 2.Reduction in protein production can be assessed by any methods known itthe art, e.g. ELISA. In certain embodiments, a puncture liver biopsysample serves as the tissue material for monitoring the reduction in theAGT gene or protein expression. In other embodiments, a blood sampleserves as the subject sample for monitoring the reduction in the agtprotein expression.

The present invention further provides methods of treatment in a subjectin need thereof, e.g., a subject diagnosed with a hypertension.

The present invention further provides methods of prophylaxis in asubject in need thereof. The treatment methods of the invention includeadministering an iRNA of the invention to a subject, e.g., a subjectthat would benefit from a reduction of AGT expression, in aprophylactically effective amount of an iRNA targeting an AGT gene or apharmaceutical composition comprising an iRNA targeting an AGT gene.

An iRNA of the invention may be administered as a “free iRNA.” A freeiRNA is administered in the absence of a pharmaceutical composition. Thenaked iRNA may be in a suitable buffer solution. The buffer solution maycomprise acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. In one embodiment, the buffer solution is phosphatebuffered saline (PBS). The pH and osmolarity of the buffer solutioncontaining the iRNA can be adjusted such that it is suitable foradministering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from an inhibition of AGT gene expressionare subjects susceptible to or diagnosed with hypertension.

In an embodiment, the method includes administering a compositionfeatured herein such that expression of the target AGT gene isdecreased, such as for about 1, 2, 3, 4, 5, 6, 1-6, 1-3, or 3-6 monthsper dose. In certain embodiments, the composition is administered onceevery 3-6 months.

Preferably, the iRNAs useful for the methods and compositions featuredherein specifically target RNAs (primary or processed) of the target AGTgene. Compositions and methods for inhibiting the expression of thesegenes using iRNAs can be prepared and performed as described herein.

Administration of the iRNA according to the methods of the invention mayresult prevention or treatment of an AGT associated disorder disorder,e.g., high blood pressure, e.g., hypertension. Diagnostic criteria forvarious types of high blood pressure are provided below.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg to about 200 mg/kg.

The iRNA is preferably administered subcutaneously, i.e., bysubcutaneous injection. One or more injections may be used to deliverthe desired dose of iRNA to a subject. The injections may be repeatedover a period of time.

The administration may be repeated on a regular basis. In certainembodiments, after an initial treatment regimen, the treatments can beadministered on a less frequent basis. A repeat-dose regimen may includeadministration of a therapeutic amount of iRNA on a regular basis, suchas once per month to once a year. In certain embodiments, the iRNA isadministered about once per month to about once every three months, orabout once every three months to about once every six months.

VIII. Diagnostic Criteria, Risk Factors, and Treatments for Hypertension

Recently practice guidelines for prevention and treatment ofhypertension were revised. Extensive reports were published by Reboussinet al. (Systematic Review for the 2017ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for thePrevention, Detection, Evaluation, and Management of High Blood Pressurein Adults: A Report of the American College of Cardiology/American HeartAssociation Task Force on Clinical Practice Guidelines. J Am CollCardiol. 2017 Nov. 7. pii: S0735-1097(17)41517-8. doi:10.1016/j.jacc.2017.11.004.) and Whelton et al. (2017ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for thePrevention, Detection, Evaluation, and Management of High Blood Pressurein Adults: A Report of the American College of Cardiology/American HeartAssociation Task Force on Clinical Practice Guidelines. J Am CollCardiol. 2017 Nov. 7. pii: S0735-1097(17)41519-1. doi:10.1016/j.jacc.2017.11.006). Some highlights of the new Guidelines areprovided below. However, the Guidelines should be understood asproviding the knowledge of those of skill in the art regardingdiagnostic and monitoring criteria and treatment for hypertension at thetime of filing of this application and are incorporated herein byreference.

A. Diagnostic Criteria

Although a continuous association exists between higher blood pressureand increased cardiovascular disease risk, it is useful to categorizeblood pressure levels for clinical and public health decision making.Blood pressure can be categorized into 4 levels on the basis of averageblood pressure measured in a healthcare setting (office pressures):normal, elevated, and stage 1 or 2 hypertension as shown in the tablebelow (from Whelton et al., 2017).

Blood Pressure Diastolic Blood Category Systolic Blood Pressure PressureNormal <120 mm Hg and <80 mm Hg Elevated 120-129 mm Hg and <80 mm HgHypertension* Stage 1 130-139 mm Hg or 80-89 mm Hg Stage 2 ≥140 mm Hg or≥90 mm Hg *Individuals with systolic blood pressure and diastolic bloodpressure in 2 categories should be designated to the higher bloodpressure category.

Blood pressure indicates blood pressure based on an average of >2careful readings obtained on ≥2 occasions. Best practices for obtainingcareful blood pressure readings are detailed in Whelton et al., 2017 andare known in the art.

This categorization differs from that previously recommended in the JNC7 report (Chobanian et al; the National High Blood Pressure EducationProgram Coordinating Committee. Seventh Report of the Joint NationalCommittee on Prevention, Detection, Evaluation, and Treatment of HighBlood Pressure. Hypertension. 2003; 42:1206-52) with stage 1hypertension now defined as a systolic blood pressure (SBP) of 130-139or a diastolic blood pressure (DBP) of 80-89 mm Hg, and with stage 2hypertension in the present document corresponding to stages 1 and 2 inthe JNC 7 report. The rationale for this categorization is based onobservational data related to the association between SBP/DBP andcardiovascular disease risk, randomized clinical trials of lifestylemodification to lower blood pressure, and randomized clinical trials oftreatment with antihypertensive medication to prevent cardiovasculardisease.

The increased risk of cardiovascular disease among adults with stage 2hypertension is well established. An increasing number of individualstudies and meta-analyses of observational data have reported a gradientof progressively higher cardiovascular disease risk going from normalblood pressure to elevated blood pressure and stage 1 hypertension. Inmany of these meta-analyses, the hazard ratios for coronary heartdisease and stroke were between 1.1 and 1.5 for the comparison ofSBP/DBP of 120-129/80-84 mm Hg versus <120/80 mm Hg and between 1.5 and2.0 for the comparison of SBP/DBP of 130-139/85-89 mm Hg versus <120/80mm Hg. This risk gradient was consistent across subgroups defined by sexand race/ethnicity. The relative increase in cardiovascular disease riskassociated with higher blood pressure was attenuated but still presentamong older adults.

Lifestyle modification and pharmacological antihypertensive treatmentare recommended for individuals with elevated blood pressure and stages1 and 2 hypertension. Clinical benefit can be obtained by a reduction ofthe stage of elevated blood pressure, even if blood pressure is notnormalized by a treatment.

B. Risk Factors

Hypertension is a complex disease that results from a combination offactors including, but not limited to, genetics, lifestyle, diet, andsecondary risk factors. Hypertension can also be associated withpregnancy. It is understood that due to the complex nature ofhypertension, it is understood that multiple interventions may berequired for treatment of hypertension. Moreover, non-pharmacologicalinterventions, including modification of diet and lifestyle, can beuseful for the prevention and treatment of hypertension. Further, anintervention may provide a clinical benefit without fully normalizingblood pressure in an individual.

1. Genetic Risk Factors

Several monogenic forms of hypertension have been identified, such asglucocorticoid-remediable aldosteronism, Liddle's syndrome, Gordon'ssyndrome, and others in which single-gene mutations fully explain thepathophysiology of hypertension, these disorders are rare. The currenttabulation of known genetic variants contributing to blood pressure andhypertension includes more than 25 rare mutations and 120 singlenucleotide polymorphisms. However, although genetic factors maycontribute to hypertension in some individuals, it is estimated thatgenetic variation accounts for only about 3.5% of blood pressurevariability.

2. Diet and Alcohol Consumption

Common environmental and lifestyle risk factors leading to hypertensioninclude poor diet, insufficient physical activity, and excess alcoholconsumption. These factors can lead to a person to become overweight orobese, further increasing the likelihood of developing or exacerbatinghypertension. Elevated blood pressure is even more strongly correlatedwith increased waist-to-hip ratio or other measures of central fatdistribution. Obesity at a young age and ongoing obesity is stronglycorrelated with hypertension later in life. Achieving a normal weightcan reduce the risk of developing high blood pressure to that of aperson who has never been obese.

Intake of sodium, potassium, magnesium, and calcium can also have asignificant effect on blood pressure. Sodium intake is positivelycorrelated with blood pressure and accounts for much of the age-relatedincrease in blood pressure. Certain groups are more sensitive toincreased sodium consumption than others including black and olderadults (≥65 years old), and those with a higher level of blood pressureor comorbidities such as chronic kidney disease, diabetes mellitus, ormetabolic syndrome. In aggregate, these groups constitute more than halfof all US adults. Salt sensitivity may be a marker for increasedcardiovascular disease and all-cause mortality, independent of bloodpressure. Currently, techniques for recognition of salt sensitivity areimpractical in a clinical setting. Therefore, salt sensitivity is bestconsidered as a group characteristic.

Potassium intake is inversely related to blood pressure and stroke, anda higher level of potassium seems to blunt the effect of sodium on bloodpressure. A lower sodium-potassium ratio is associated with a lowerblood pressure than that noted for corresponding levels of sodium orpotassium on their own. A similar observation has been made for risk ofcardiovascular disease.

Alcohol consumption has long been associated with high blood pressure.In the US, it has been estimated that alcohol consumption accounts forabout 10% of the population burden of hypertension, with the burdenbeing greater in men than women.

It is understood that changes in diet or alcohol consumption can be anaspect of prevention or treatment of hypertension.

3. Physical Activity

There is a well-established inverse correlation between physicalactivity/physical fitness and blood pressure levels. Even modest levelsof physical activity have been demonstrated to be beneficial indecreasing hypertension.

It is understood that an increase in physical activity can be an aspectof prevention or treatment of hypertension.

4. Secondary Risk Factors

Secondary hypertension can underlie severe elevation of blood pressure,pharmacologically resistant hypertension, sudden onset of hypertension,increased blood pressure in patients with hypertension previouslycontrolled on drug therapy, onset of diastolic hypertension in olderadults, and target organ damage disproportionate to the duration orseverity of the hypertension. Although secondary hypertension should besuspected in younger patients (<30 years of age) with elevated bloodpressure, it is not uncommon for primary hypertension to manifest at ayounger age, especially in blacks, and some forms of secondaryhypertension, such as renovascular disease, are more common at older age(≥65 years of age). Many of the causes of secondary hypertension arestrongly associated with clinical findings or groups of findings thatsuggest a specific disorder. In such cases, treatment of the underlyingcondition may resolve the findings of elevated blood pressure withoutadministering agents typically used for the treatment of hypertension.

5. Pregnancy

Pregnancy is a risk factor for high blood pressure, and high bloodpressure during pregnancy is a risk factor for cardiovascular diseaseand hypertension later in life. A Report on pregnancy associatedhypertension was published in 2013 by the American College of Obstetricsand Gynecology (ACOG) (American College of Obstetricians andGynecologists, Task Force on Hypertension in Pregnancy. Hypertension inpregnancy. Report of the American College of Obstetricians andGynecologists' Task Force on Hypertension in Pregnancy. Obstet Gynecol.2013; 122:1122-31). Some highlights of the Report are provided below.However, the Report should be understood as providing the knowledge ofthose of skill in the art regarding diagnostic and monitoring criteriaand treatment for hypertension in pregnancy at the time of filing ofthis application and are incorporated herein by reference.′

The diagnostic criteria for preeclampsia are provided in the table below(from Table 1 of the ACOG report, 2013).

Blood Pressure ≥140 mm Hg diastolic or ≥90 mm Hg diastolic on twooccasions at least 4 hours apart after 20 weeks of gestation in a womanwith a previously normal blood pressure ≥160 mm Hg systolic or ≥110 mmHg diastolic, hypertension can be confirmed within a short interval(minutes) to facilitate timely antihypertensive therapy and Proteinurea≥300 mg per 24-hour urine collection (or this amount extrapolated for atimed collection) Or Protein/ creatinine ratio ≥0.3 (each measured asmg/dL) Or in the absence of proteinurea, new onset of hypertension withthe new onset of an of the following: Thrombocytopenia Platelet count≤100,000/microliter Renal insufficiency Serum creatinine concentration≥1.1 mg/dL or a doubling of the serum creatinine concentration in theabsence of other renal disease Impaired liver Elevated bloodconcentrations if liver function transaminases to twice normalconcentration Pulmonary edema Cerebral or visual symptoms

Blood Pressure management during pregnancy is complicated by the factthat many commonly used antihypertensive agents, including ACEinhibitors and ARBs, are contraindicated during pregnancy because ofpotential harm to the fetus. The goal of antihypertensive treatmentduring pregnancy includes prevention of severe hypertension and thepossibility of prolonging gestation to allow the fetus more time tomature before delivery. A review of treatment for pregnancy-associatedsevere hypertension found insufficient evidence to recommend specificagents; rather, clinician experience was recommended in this setting(Duley L, Meher S, Jones L. Drugs for treatment of very high bloodpressure during pregnancy. Cochrane Database Syst Rev. 2013;7:CD001449).

C. Treatments

Treatment of high blood pressure is complex as it is frequently presentwith other comorbidities, often including reduced renal function, forwhich the subject may also be undergoing treatment. Clinicians managingadults with high blood pressure should focus on overall patient health,with a particular emphasis on reducing the risk of future adversecardiovascular disease outcomes. All patient risk factors need to bemanaged in an integrated fashion with a comprehensive set ofnonpharmacological and pharmacological strategies. As patient bloodpressure and risk of future cardiovascular disease events increase,blood pressure management should be intensified.

Whereas treatment of high blood pressure with blood pressure-loweringmedications on the basis of blood pressure level alone is consideredcost effective, use of a combination of absolute cardiovascular diseaserisk and blood pressure level to guide such treatment is more efficientand cost effective at reducing risk of cardiovascular disease than isuse of blood pressure level alone. Many patients started on a singleagent will subsequently require ≥2 drugs from different pharmacologicalclasses to reach their blood pressure goals. Knowledge of thepharmacological mechanisms of action of each agent is important. Drugregimens with complementary activity, where a second antihypertensiveagent is used to block compensatory responses to the initial agent oraffect a different pressor mechanism, can result in additive lowering ofblood pressure. For example, thiazide diuretics may stimulate therenin-angiotensin-aldosterone system. By adding an ACE inhibitor or ARBto the thiazide, an additive blood pressure lowering effect may beobtained. Use of combination therapy may also improve adherence. Several2- and 3-fixed-dose drug combinations of antihypertensive drug therapyare available, with complementary mechanisms of action among thecomponents.

Table 18 from Whelton et al. 2017 listing oral antihypertensive drugs isprovided below. Classes of therapeutic agents for the treatment of highblood pressure and drugs that fall within those classes are provided.Dose ranges, frequencies, and comments are also provided.

Usual Dose, Daily Range Fre- Class Drug (mg/d)* quency Comments Primaryagents Thiazide Chlorthalidone 12.5-25 1 Chlorthalidone is or thiazide-Hydro- 25-50 1 preferred on the basis of type diuretics chlorothiazideprolonged half-life and proven Indapamide 1.25-2.5 1 trial reduction ofCVD. Metolazone 2.5-10 1 Monitor for hyponatremia and hypokalemia, uricacid and calcium levels. Use with caution in patients with history ofacute gout unless patient is on uric acid-lowering therapy. ACEinhibitors Benazepril 10-40 1 or 2 Do not use in Captopril 12.5-150 2 or3 combination with ARBs or Enalapril 5-40 1 or 2 direct renin inhibitorFosinopril 10-40 1 There is an increased Lisinopril 10-40 1 risk ofhyperkalemia, Moexipril 7.5-30 1 or 2 especially in patents with CKDPerindopril 4-16 1 or in those on K⁺ supplements Quinapril 10-80 1 or 2or K⁺-sparing drugs. Ramipril 2.5-10 1 or 2 There is a risk of acuteTrandolapril 1-4 1 renal failure in patients with severe bilateral renalartery stenosis. Do no use if patient has history of angioedema with ACEinhibitors. Avoid in pregnancy. ARBs Azilsartan 40-80 1 Do not use inCandesartan 8-32 1 combination with ACE Eprosartan 600-800 1 or 2inhibitors or direct renin Irbesartan 150-300 1 inhibitors. Losartan50-100 1 or 2 There is an increased Olmesartan 20-40 1 risk ofhyperkalemia in CKD or Telmisartan 20-80 1 in those on K⁺ supplements orValsartan 80-320 1 K⁺-sparing drugs. There is a risk of acute renalfailure in patients with severe bilateral renal artery stenosis. Do notuse if patient has history of angioedema with ARBs. Patients with ahistory of angioedema with an ACE inhibitor can receive an ARB beginning6 weeks after ACE inhibitor is discontinued. Avoid in pregnancy. CCB-Amlodipine 2.5-10 1 Avoid use in patients dihydropyridines Felodipine5-10 1 with HFrEF; amlodipine or Isradipine 5-10 2 felodipine may beused if Nicardipine SR 5-20 1 required Nifedipine LA 60-120 1 They areassociated Nisoldipine 30-90 1 with dose-related pedal edema, which ismore common in women than men. CCB- Diltiazem SR 180-360 2 Avoid routineuse with nondihydropyridines Diltiazem ER 120-480 1 beta blockersbecause of Verapamil IR 40-80 3 increased risk of bradycardia VerapamilSR 120-480 1 or 2 and heart block. Verapamil- 100-480 1 (in the Do notuse in patients delayed evening) with HFrEF. onset ER There are drug(various interactions with diltiazem and forms) verapamil (CYP3A4 majorsubstrate and moderate inhibitor). Secondary agents Diuretics-loopBumetanide 0.5-4 2 There are preferred Furosemide 20-80 2 diuretics inpatients with Torsemide 5-10 1 symptomatic HF. They are preferred overthiazides in patients with moderate-to- severe CKD (e.g., GFR <30mL/min). Diuretics-potassium Amiloride 5-10 1 or 2 These are sparingmonotherapy agents and minimally effective antihypertensive agents.Triamterene 50-100 1 or 2 Combination therapy of potassium-sparingdiuretic with a thiazide can be considered in patients with hypokalemiaon thiazide monotherapy. Avoid in patients with significate CKD (e.g.GFR <45 mL/min). Diuretics- Eplerenone 50-100 12 These are preferredaldosterone Spironolactone 25-100 1 agents in primary aldosteronismantagonists and resistant hypertension. Spironolactone is associatedwith greater risk of gynecomastia and impotence as compared witheplerenone. This is common add- on therapy in resistant hypertension.Avoid use with K⁺ supplements, other K⁺-sparing diuretics, orsignificant renal dysfunction. Eplerenone often requires twice-dailydosing for adequate BP lowering. Beta blockers- Atenolol 25-100 12 Betablockers are not cardioselective Betaxolol 5-20 1 recommended asfirst-line Bisoprolol 2.5-10 1 agents unless the patient has Metoprolol100-400 2 IHD or HF. tartrate These are preferred in Metoprolol 50-200 1patients with broncho spastic succinate airway disease requiring a betablocker. Bisoprolol and metoprolol succinate are preferred in patientswith HFrEF. Avoid abrupt cessation. Beta blockers- Nebivolol 5-40 1Nebivolol induces cardioselective and nitric oxide-inducesd vasodilatoryvasodilation. Avoid abrupt cessation. Beta blockers- Nadolol 40-120 1Avoid in patients with noncardioselective Propanolol IR 160-480 2reactive ariways disease. Propanolol LA 80-320 1 Avoid abrupt cessation.Beta blockers- Acebutolol 200-800 1 Generally avoid, intrinsic Carteolol2.5-10 1 especially in patients with IHD sympathomimetic Penbutolol10-40 1 or HF. activity Pindolol 10-60 2 Avoid abrupt cessation. Betablockers- Carvediol 12.5-50 2 Carvedilol is preferred combined alpha-andCarvedilol 20-80 1 in patients with HFrEF. beta receptor phosphate Avoidabrupt Labetalol 200-800 2 cessation. Direct renin Aliskiren 150-300 1Do not use in inhibitor combination with ACE inhibitors or ARBs.Aliskiren is very long acting. There is an increased risk ofhyperkalemina in CKD or in those on K⁺ supplements or K⁺-sparring drugs.Aliskiren may cause acute renal failure in patients with severebilateral renal artery stenosis. Avoid in pregnacy. Alpha-1-blockersDoxazosin 1-8 1 These are associated Prazosin 2-20 2 or 3 withorthostatic hypotension, Terazosin 1-20 1 or 2 especially in olderadults. They may be considered as second-line agent in patients withconcominant BPH. Central alpha- Clonidine oral 0.1-0.8 2 These aregenerally agonist and other Clonidine patch 0.1-0.3 1 weekly reserved aslast-line because of centrally acting Methyldopa 250-1000 2 significantCNS adverse drugs Guanfacine 0.5-2 1 effects, especially in olderadults. Avoid abrupt discontinuation of clonidine, which may inducehypertensive crisis; clonidine must be tapered to avoid reboundhypertension. Direct vasodilators Hydrazine 250-200 2 or 3 These areassociated Minoxidil 5-100 1-3 with sodium and water retention andreflex tachycardia; use with a diuretic and beta blocker. Hydralazine isassociated with drug-induced lupus-like syndrome at higher doses.Minoxidil is associated with hirsutism and required a loop diurestic.Minoxidil can induce pericardial effusion. *Dosages may vary from thoselisted in the FDA approved labeling (available athtttps://dailymed.nlm.nih.gov/dailymed/). ACE indicatesangiotensin-converting enzyme; ARB, angiotensin receptor blocker; BP,blood pressure; BPH, benign prostatic hyperplasia; CCB, calcium channelblocker; CKD, chronic kidney disease; CNS, central nervous system; CVD,cardiovascular disease; ER, extended release; GFR, glomerular filtrationrate; HF, heart failure; HFrEF, heart failure with reduced ejectionfraction; IHD, ischemic heart disease; IR, immediate release; LA,long-acting; and SR, sustained release. From, Chobanian et al. (2003)The JNC 7 Report. JAMA 289(19):2560.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Sequence Listing, are herebyincorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained 20 from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Design

A set of siRNAs targeting the human AGT gene (human: NCBI refseqIDNM_000029.3; NCBI GeneID: 183) was designed using custom R and Pythonscripts. The human NM_000029 REFSEQ mRNA, version 3, has a length of2587 bases.

A detailed list of the unmodified AGT sense and antisense strandnucleotide sequences is shown in Table 3. A detailed list of themodified AGT sense and antisense strand nucleotide sequences is shown inTable 5.

siRNA Synthesis siRNAs were synthesized and annealed using routinemethods known in the art.

Example 2. In Vitro Screening Methods

Cell Culture and 384-Well Transfections

Hep3b cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium(Gibco) supplemented with 10% FBS (ATCC) before being released from theplate by trypsinization.

Transfection was performed by adding 4.9 μl of Opti-MEM plus 0.1 μl ofLipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) to 5 μl of each siRNA duplex to an individual well in a384-well plate. The mixture was then incubated at room temperature for20 minutes. Firty μl of complete growth media containing 5,000 Hep3bcells were then added to the siRNA mixture. Cells were incubated for 24hours prior to RNA purification. Single dose experiments were performedat 10 nM and 0.1 nM final duplex concentration, and dose responseexperiments were performed using an eight-point six-fold serial dilutionover the range of 10 nM to 37.5 fM.

Additional dsRNA agents targeting an AGT mRNA are described in PCTPublication No. WO 2015/179724, the entire contents of which areincorporated herein by reference.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen™,Part #: 610-12)

Cells were lysed in 75 μl of Lysis/Binding Buffer containing 3 uL ofbeads per well and mixed for 10 minutes on an electrostatic shaker. Thewashing steps were automated on a Biotek EL406, using a magnetic platesupport. Beads were washed (in 90 μL) once in Buffer A, once in BufferB, and twice in Buffer E, with aspiration steps in between. Following afinal aspiration, complete 10 μL RT mixture was added to each well, asdescribed below.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 1 ul 10× Buffer, 0.4 μl 25×dNTPs, 1 μl Random primers,0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H₂Oper reaction were added per well. Plates were sealed, agitated for 10minutes on an electrostatic shaker, and then incubated at 37 degrees C.for 2 hours. Following this, the plates were agitated at 80 degrees C.for 8 minutes.

Real Time PCR:

Two μl of cDNA were added to a master mix containing 0.5 μl of humanGAPDH TaqMan Probe (4326317E), 0.5 μl human AGT (Hs00174854 ml), 2 μlnuclease-free water and 5 μl Lightcycler 480 probe master mix (Roche Cat#04887301001) per well in a 384 well plates (Roche cat #04887301001).Real time PCR was done in a LightCycler480 Real Time PCR system (Roche).

To calculate relative fold change, data were analyzed using the ΔΔCtmethod and normalized to assays performed with cells transfected with 10nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4parameter fit model using XLFit and normalized to cells transfected withAD-1955 or mock-transfected. The sense and antisense sequences ofAD-1955 are: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO:19) andantisense UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 20). Results from thescreening are shown in Table 4.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3'-phosphate Ab beta-L-adenosine-3{grave over ( )}-phosphateAbs beta-L-adenosine-3{grave over ( )}-phosphorothioate Af2'-fluoroadenosine-3'-phosphate Afs2'-fluoroadenosine-3'-phosphorothioate As adenosine-3'-phosphorothioateC cytidine-3'-phosphate Cb beta-L-cytidine-3{grave over ( )}-phosphateCbs beta-L-cytidine-3'-phosphorothioate Cf2'-fluorocytidine-3'-phosphate Cfs 2'-fluorocytidine-3'-phosphorothioateCs cytidine-3'-phosphorothioate G guanosine-3'-phosphate Gbbeta-L-guanosine-3{grave over ( )}-phosphate Gbsbeta-L-guanosine-3{grave over ( )}-phosphorothioate Gf2'-fluoroguanosine-3'-phosphate Gfs2'-fluoroguanosine-3'-phosphorothioate Gs guanosine-3'-phosphorothioateT 5'-me thyluri dine-3'-phosphate Tf2'-fluoro-5-methyluridine-3'-phosphate Tfs2'-fluoro-5-methyluridine-3'-phosphorothioate Ts5-methyluridine-3'-phosphorothioate U Uridine-3'-phosphate Uf2'-fluorouridine-3'-phosphate Ufs 2'-fluorouridine-3'-phosphorothioateUs uridine-3'-phosphorothioate N any nucleotide, modified or unmodifieda 2′-O-methyladenosine-3'-phosphate as2′-O-methyladenosine-3'-phosphorothioate c2′-O-methylcytidine-3'-phosphate cs2′-O-methylcytidine-3'-phosphorothioate g2′-O-methylguanosine-3'-phosphate gs2′-O-methylguanosine-3'-phosphorothioate t2'-O-methyl-5-methyluridine-3'-phosphate ts2'-O-methyl-5-methyluridine-3'-phosphorothioate u2′-O-methyluridine-3'-phosphate us2′-O-methyluridine-3'-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]- 4-hydroxyprolinol(Hyp-(GalNAc-alkyl)3) Y34 2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic 2′-OMe furanose) Y44 inverted abasic DNA(2-hydroxymethyl- tetrahydrofurane-5-phosphate) (Agn) Adenosine-glycolnucleic acid (GNA) (Cgn) Cytidine-glycol nucleic acid (GNA) (Ggn)Guanosine-glycol nucleic acid (GNA) (Tgn) Thymidine-glycol nucleic acid(GNA) S-Isomer P Phosphate VP Vinyl-phosphate (Aam) 2{grave over( )}-O-(N-methylacetamide)adenosine-3{grave over ( )}-phosphate (Aams)2{grave over ( )}-O-(N-methylacetamide)adenosine-3{grave over( )}-phosphorothioate (Gam) 2{grave over( )}-O-(N-methylacetamide)guanosine-3{grave over ( )}-phosphate (Gams)2{grave over ( )}-O-(N-methylacetamide)guanosine-3{grave over( )}-phosphorothioate (Tam) 2{grave over( )}-O-(N-methylacetamide)thymidine-3{grave over ( )}-phosphate (Tams)2{grave over ( )}-O-(N-methylace tamide)thymidine-3{grave over( )}-phosphorothioate dA 2{grave over ( )}-deoxyadenosine-3{grave over( )}-phosphate dAs 2{grave over ( )}-deoxyadenosine-3{grave over( )}-phosphorothioate dC 2{grave over ( )}-deoxycytidine-3{grave over( )}-phosphate dCs 2{grave over ( )}-deoxycytidine-3{grave over( )}-phosphorothioate dG 2{grave over ( )}-deoxyguanosine-3{grave over( )}-phosphate dGs 2{grave over ( )}-deoxyguanosine-3{grave over( )}-phosphorothioate dT 2{grave over ( )}-deoxythymidine-3{grave over( )}-phosphate dTs 2{grave over ( )}-deoxythymidine-3{grave over( )}-phosphorothioate dU 2{grave over ( )}-deoxyuridine dUs 2{grave over( )}-deoxyuridine-3{grave over ( )}-phosphorothioate (Aeo) 2{grave over( )}-O-methoxyethyladenosine-3{grave over ( )}-phosphate (Aeos) 2{graveover ( )}-O-methoxyethyladenosine-3{grave over ( )}-phosphorothioate(Geo) 2{grave over ( )}-O-methoxyethylguanosine-3{grave over( )}-phosphate (Geos) 2{grave over ( )}-O-methoxyethylguanosine-3{graveover ( )}-phosphorothioate (Teo) 2{grave over( )}-O-methoxyethyl-5-methyluridine-3{grave over ( )}-phosphate (Teos)2{grave over ( )}-O-methoxyethyl-5-methyluridine-3{grave over( )}-phosphorothioate (m5Ceo) 2{grave over( )}-O-methoxyethyl-5-methylcytidine-3{grave over ( )}-phosphate(m5Ceos) 2{grave over ( )}-O-methoxyethyl-5-methylcytidine-3{grave over( )}-phosphorothioate (A3m) 3{grave over ( )}-O-methyladeno sine-2{graveover ( )}-phosphate (A3mx) 3{grave over ( )}-O-methyl-xylofuranosyladenosine-2{grave over ( )}-phosphate (G3m) 3{grave over( )}-O-methylguanosine-2{grave over ( )}-phosphate (G3mx) 3{grave over( )}-O-methyl-xylofuranosylguanosine-2{grave over ( )}-phosphate (C3m)3{grave over ( )}-O-methylcytidine-2{grave over ( )}-phosphate (C3mx)3{grave over ( )}-O-methyl-xylofuranosylcytidine-2{grave over( )}-phosphate (U3m) 3{grave over ( )}-O-methyluridine-2{grave over( )}-phosphate U3mx) 3{grave over( )}-O-methyl-xylofuranosyluridine-2{grave over ( )}-phosphate (m5Cam)2{grave over ( )}-O-(N-methylace tamide)-5-methylcytidine-3{grave over( )}-phosphate (m5Cams) 2{grave over ( )}-O-(N-methylace tamide)-5-methylcytidine-3{grave over ( )}-phosphorothioate (Chd)2′-O-hexadecyl-cytidine-3′-phosphate (Chds)2′-O-hexadecyl-cytidine-3′-phosphorothioate (Uhd)2′-O-hexadecyl-uridine-3′-phosphate (Uhds)2′-O-hexadecyl-uridine-3′-phosphorothioate (pshe)Hydroxyethylphosphorothioate

TABLE 3Unmodified Sense and Antisense Strand Sequences of AGT dsRNA AgentsSense SEQ Antisense SEQ Duplex Oligo Sense ID Range in Oligo AntisenseID Range in Name Name Sequence 5′ to 3′ NO: NM_000029.3 NameSequence 5′ to 3′ NO: NM_000029.3_ AD-84704 A-168477CACAAUGAGAGUACCUGUGAA 21 644-664 A-168478 UUCACAGGUACUCUCAUUGUGGA 205642-664 AD-84705 A-168479 GUCUCCCACCUUUUCUUCUAA 22 2076-2096 A-168480UUAGAAGAAAAGGUGGGAGACUG 206 2074-2096 AD-84706 A-168481ACUUUCCAGCAAAACUCCCUA 23 1586-1606 A-168482 UAGGGAGUUUUGCUGGAAAGUGA 2071584-1606 AD-84707 A-168483 CCUCAACUGGAUGAAGAAACU 24 1603-1623 A-168484AGUUUCUUCAUCCAGUUGAGGGA 208 1601-1623 AD-84708 A-168485CUGUUUGCUGUGUAUGAUCAA 25 1889-1909 A-168486 UUGAUCAUACACAGCAAACAGGA 2091887-1909 AD-84709 A-168487 UUUGCUGUGUAUGAUCAAAGA 26 1892-1912 A-168488UCUUUGAUCAUACACAGCAAACA 210 1890-1912 AD-84710 A-168489CCGACCAGCUUGUUUGUGAAA 27 2283-2303 A-168490 UUUCACAAACAAGCUGGUCGGUU 2112281-2303 AD-84711 A-168491 UCCAACCGACCAGCUUGUUUA 28 2278-2298 A-168492UAAACAAGCUGGUCGGUUGGAAU 212 2276-2298 AD-84712 A-168493CCAUUCCUGUUUGCUGUGUAU 29 1883-1903 A-168494 AUACACAGCAAACAGGAAUGGGC 2131881-1903 AD-84713 A-168495 CACCUUUUCUUCUAAUGAGUA 30 2082-2102 A-168496UACUCAUUAGAAGAAAAGGUGGG 214 2080-2102 AD-84714 A-168497GUUUGCUGUGUAUGAUCAAAA 31 1891-1911 A-168498 UUUUGAUCAUACACAGCAAACAG 2151889-1911 AD-84715 A-168499 GCUGAGAAGAUUGACAGGUUA 32 1250-1270 A-168500UAACCUGUCAAUCUUCUCAGCAG 216 1248-1270 AD-84716 A-168501UUCCAGCAAAACUCCCUCAAA 33 1589-1609 A-168502 UUUGAGGGAGUUUUGCUGGAAAG 2171587-1609 AD-84717 A-168503 UGCUGAGAAGAUUGACAGGUU 34 1249-1269 A-168504AACCUGUCAAUCUUCUCAGCAGC 218 1247-1269 AD-84718 A-168505UCUCACUUUCCAGCAAAACUA 35 1582-1602 A-168506 UAGUUUUGCUGGAAAGUGAGACC 2191580-1602 AD-84719 A-168507 UCCACAAUGAGAGUACCUGUA 36 642-662 A-168508UACAGGUACUCUCAUUGUGGAUG 220 640-662 AD-84720 A-168509CCACCUCGUCAUCCACAAUGA 37 631-651 A-168510 UCAUUGUGGAUGACGAGGUGGAA 221629-651 AD-84721 A-168511 UCACUUUCCAGCAAAACUCCA 38 1584-1604 A-168512UGGAGUUUUGCUGGAAAGUGAGA 222 1582-1604 AD-84722 A-168513UCCCUCAACUGGAUGAAGAAA 39 1601-1621 A-168514 UUUCUUCAUCCAGUUGAGGGAGU 2231599-1621 AD-84723 A-168515 GAGAGUACCUGUGAGCAGCUA 40 650-670 A-168516UAGCUGCUCACAGGUACUCUCAU 224 648-670 AD-84724 A-168517AGAAUUCCAACCGACCAGCUU 41 2273-2293 A-168518 AAGCUGGUCGGUUGGAAUUCUUU 2252271-2293 AD-84725 A-168519 CAUUCCUGUUUGCUGUGUAUA 42 1884-1904 A-168520UAUACACAGCAAACAGGAAUGGG 226 1882-1904 AD-84726 A-168521GAAUUCCAACCGACCAGCUUA 43 2274-2294 A-168522 UAAGCUGGUCGGUUGGAAUUCUU 2272272-2294 AD-84727 A-168523 CAUCCACAAUGAGAGUACCUA 44 640-660 A-168524UAGGUACUCUCAUUGUGGAUGAC 228 638-660 AD-84728 A-168525CCCAUUCCUGUUUGCUGUGUA 45 1882-1902 A-168526 UACACAGCAAACAGGAAUGGGCG 2291880-1902 AD-84729 A-168527 CUGGGUUUAUUUUAGAGAAUA 46 2202-2222 A-168528UAUUCUCUAAAAUAAACCCAGCA 230 2200-2222 AD-84730 A-168529GCUGGGUUUAUUUUAGAGAAU 47 2201-2221 A-168530 AUUCUCUAAAAUAAACCCAGCAA 2312199-2221 AD-84731 A-168531 AUGGCAUGCACAGUGAGCUAU 48 861-881 A-168532AUAGCUCACUGUGCAUGCCAUAU 232 859-881 AD-84732 A-168533GAGAGAGCCCACAGAGUCUAA 49 1816-1836 A-168534 UUAGACUCUGUGGGCUCUCUCUC 2331814-1836 AD-84733 A-168535 GCAAGAACCAGUGUUUAGCGA 50 2234-2254 A-168536UCGCUAAACACUGGUUCUUGCCU 234 2232-2254 AD-84734 A-168537CCAGCAAAACUCCCUCAACUA 51 1591-1611 A-168538 UAGUUGAGGGAGUUUUGCUGGAA 2351589-1611 AD-84735 A-168539 CACCUCGUCAUCCACAAUGAA 52 632-652 A-168540UUCAUUGUGGAUGACGAGGUGGA 236 630-652 AD-84736 A-168541CGUCAUCCACAAUGAGAGUAA 53 637-657 A-168542 UUACUCUCAUUGUGGAUGACGAG 237635-657 AD-84737 A-168543 CUCCCACGCUCUCUGGACUUA 54 1211-1231 A-168544UAAGUCCAGAGAGCGUGGGAGGA 238 1209-1231 AD-84738 A-168545AACUCCCUCAACUGGAUGAAA 55 1598-1618 A-168546 UUUCAUCCAGUUGAGGGAGUUUU 2391596-1618 AD-84739 A-168547 UGAGAAGAUUGACAGGUUCAU 56 1252-1272 A-168548AUGAACCUGUCAAUCUUCUCAGC 240 1250-1272 AD-84740 A-168549CCUCGUCAUCCACAAUGAGAA 57 634-654 A-168550 UUCUCAUUGUGGAUGACGAGGUG 241632-654 AD-84741 A-168551 GAGAAGAUUGACAGGUUCAUA 58 1253-1273 A-168552UAUGAACCUGUCAAUCUUCUCAG 242 1251-1273 AD-84742 A-168553CUUCUUGGGCUUCCGUAUAUA 59 841-861 A-168554 UAUAUACGGAAGCCCAAGAAGUU 243839-861 AD-84743 A-168555 UCCACCUCGUCAUCCACAAUA 60 630-650 A-168556UAUUGUGGAUGACGAGGUGGAAG 244 628-650 AD-84744 A-168557AGAUUGACAGGUUCAUGCAGA 61 1257-1277 A-168558 UCUGCAUGAACCUGUCAAUCUUC 2451255-1277 AD-84745 A-168559 CUCCCUCAACUGGAUGAAGAA 62 1600-1620 A-168560UUCUUCAUCCAGUUGAGGGAGUU 246 1598-1620 AD-84746 A-168561AAUGAGAGUACCUGUGAGCAA 63 647-667 A-168562 UUGCUCACAGGUACUCUCAUUGU 247645-667 AD-85431 A-168469 CCACCUUUUCUUCUAAUGAGU 64 2081-2101 A-170464ACUCAUUAGAAGAAAAGGUGGGA 248 2079-2101 AD-85432 A-168471CGACCAGCUUGUUUGUGAAAA 65 2284-2304 A-170465 UUUUCACAAACAAGCUGGUCGGU 2492282-2304 AD-85433 A-168473 ACCUUUUCUUCUAAUGAGUCA 66 2083-2103 A-170466UGACUCAUUAGAAGAAAAGGUGG 250 2081-2103 AD-85434 A-168475GUCAUCCACAAUGAGAGUACA 67 638-658 A-170467 UGUACUCUCAUUGUGGAUGACGA 251636-658 AD-85435 A-168477 CACAAUGAGAGUACCUGUGAA 68 644-664 A-170468UUCACAGGUACUCUCAUUGUGGA 252 642-664 AD-85436 A-168479GUCUCCCACCUUUUCUUCUAA 69 2076-2096 A-170469 UUAGAAGAAAAGGUGGGAGACUG 2532074-2096 AD-85437 A-168481 ACUUUCCAGCAAAACUCCCUA 70 1586-1606 A-170470UAGGGAGUUUUGCUGGAAAGUGA 254 1584-1606 AD-85438 A-168483CCUCAACUGGAUGAAGAAACU 71 1603-1623 A-170471 AGUUUCUUCAUCCAGUUGAGGGA 2551601-1623 AD-85439 A-168485 CUGUUUGCUGUGUAUGAUCAA 72 1889-1909 A-170472UUGAUCAUACACAGCAAACAGGA 256 1887-1909 AD-85440 A-168487UUUGCUGUGUAUGAUCAAAGA 73 1892-1912 A-170473 UCUUUGAUCAUACACAGCAAACA 2571890-1912 AD-85441 A-168489 CCGACCAGCUUGUUUGUGAAA 74 2283-2303 A-170474UUUCACAAACAAGCUGGUCGGUU 258 2281-2303 AD-85442 A-168491UCCAACCGACCAGCUUGUUUA 75 2278-2298 A-170475 UAAACAAGCUGGUCGGUUGGAAU 2592276-2298 AD-85443 A-168493 CCAUUCCUGUUUGCUGUGUAU 76 1883-1903 A-170476AUACACAGCAAACAGGAAUGGGC 260 1881-1903 AD-85444 A-168495CACCUUUUCUUCUAAUGAGUA 77 2082-2102 A-170477 UACUCAUUAGAAGAAAAGGUGGG 2612080-2102 AD-85445 A-168497 GUUUGCUGUGUAUGAUCAAAA 78 1891-1911 A-170478UUUUGAUCAUACACAGCAAACAG 262 1889-1911 AD-85446 A-168499GCUGAGAAGAUUGACAGGUUA 79 1250-1270 A-170479 UAACCUGUCAAUCUUCUCAGCAG 2631248-1270 AD-85447 A-168501 UUCCAGCAAAACUCCCUCAAA 80 1589-1609 A-170480UUUGAGGGAGUUUUGCUGGAAAG 264 1587-1609 AD-85448 A-168503UGCUGAGAAGAUUGACAGGUU 81 1249-1269 A-170481 AACCUGUCAAUCUUCUCAGCAGC 2651247-1269 AD-85449 A-168505 UCUCACUUUCCAGCAAAACUA 82 1582-1602 A-170482UAGUUUUGCUGGAAAGUGAGACC 266 1580-1602 AD-85450 A-168507UCCACAAUGAGAGUACCUGUA 83 642-662 A-170483 UACAGGUACUCUCAUUGUGGAUG 267640-662 AD-85451 A-168509 CCACCUCGUCAUCCACAAUGA 84 631-651 A-170484UCAUUGUGGAUGACGAGGUGGAA 268 629-651 AD-85452 A-168511UCACUUUCCAGCAAAACUCCA 85 1584-1604 A-170485 UGGAGUUUUGCUGGAAAGUGAGA 2691582-1604 AD-85453 A-168513 UCCCUCAACUGGAUGAAGAAA 86 1601-1621 A-170486UUUCUUCAUCCAGUUGAGGGAGU 270 1599-1621 AD-85454 A-168515GAGAGUACCUGUGAGCAGCUA 87 650-670 A-170487 UAGCUGCUCACAGGUACUCUCAU 271648-670 AD-85455 A-168517 AGAAUUCCAACCGACCAGCUU 88 2273-2293 A-170488AAGCUGGUCGGUUGGAAUUCUUU 272 2271-2293 AD-85456 A-168519CAUUCCUGUUUGCUGUGUAUA 89 1884-1904 A-170489 UAUACACAGCAAACAGGAAUGGG 2731882-1904 AD-85457 A-168521 GAAUUCCAACCGACCAGCUUA 90 2274-2294 A-170490UAAGCUGGUCGGUUGGAAUUCUU 274 2272-2294 AD-85458 A-168523CAUCCACAAUGAGAGUACCUA 91 640-660 A-170491 UAGGUACUCUCAUUGUGGAUGAC 275638-660 AD-85459 A-168525 CCCAUUCCUGUUUGCUGUGUA 92 1882-1902 A-170492UACACAGCAAACAGGAAUGGGCG 276 1880-1902 AD-85460 A-168527CUGGGUUUAUUUUAGAGAAUA 93 2202-2222 A-170493 UAUUCUCUAAAAUAAACCCAGCA 2772200-2222 AD-85461 A-168529 GCUGGGUUUAUUUUAGAGAAU 94 2201-2221 A-170494AUUCUCUAAAAUAAACCCAGCAA 278 2199-2221 AD-85462 A-168531AUGGCAUGCACAGUGAGCUAU 95 861-881 A-170495 AUAGCUCACUGUGCAUGCCAUAU 279859-881 AD-85463 A-168533 GAGAGAGCCCACAGAGUCUAA 96 1816-1836 A-170496UUAGACUCUGUGGGCUCUCUCUC 280 1814-1836 AD-85464 A-168535GCAAGAACCAGUGUUUAGCGA 97 2234-2254 A-170497 UCGCUAAACACUGGUUCUUGCCU 2812232-2254 AD-85465 A-168537 CCAGCAAAACUCCCUCAACUA 98 1591-1611 A-170498UAGUUGAGGGAGUUUUGCUGGAA 282 1589-1611 AD-85466 A-168539CACCUCGUCAUCCACAAUGAA 99 632-652 A-170499 UUCAUUGUGGAUGACGAGGUGGA 283630-652 AD-85467 A-168541 CGUCAUCCACAAUGAGAGUAA 100 637-657 A-170500UUACUCUCAUUGUGGAUGACGAG 284 635-657 AD-85468 A-168543CUCCCACGCUCUCUGGACUUA 101 1211-1231 A-170501 UAAGUCCAGAGAGCGUGGGAGGA 2851209-1231 AD-85469 A-168545 AACUCCCUCAACUGGAUGAAA 102 1598-1618 A-170502UUUCAUCCAGUUGAGGGAGUUUU 286 1596-1618 AD-85470 A-168547UGAGAAGAUUGACAGGUUCAU 103 1252-1272 A-170503 AUGAACCUGUCAAUCUUCUCAGC 2871250-1272 AD-85471 A-168549 CCUCGUCAUCCACAAUGAGAA 104 634-654 A-170504UUCUCAUUGUGGAUGACGAGGUG 288 632-654 AD-85472 A-168551GAGAAGAUUGACAGGUUCAUA 105 1253-1273 A-170505 UAUGAACCUGUCAAUCUUCUCAG 2891251-1273 AD-85473 A-168553 CUUCUUGGGCUUCCGUAUAUA 106 841-861 A-170506UAUAUACGGAAGCCCAAGAAGUU 290 839-861 AD-85474 A-168555UCCACCUCGUCAUCCACAAUA 107 630-650 A-170507 UAUUGUGGAUGACGAGGUGGAAG 291628-650 AD-85475 A-168557 AGAUUGACAGGUUCAUGCAGA 108 1257-1277 A-170508UCUGCAUGAACCUGUCAAUCUUC 292 1255-1277 AD-85476 A-168559CUCCCUCAACUGGAUGAAGAA 109 1600-1620 A-170509 UUCUUCAUCCAGUUGAGGGAGUU 2931598-1620 AD-85477 A-168561 AAUGAGAGUACCUGUGAGCAA 110 647-667 A-170510UUGCUCACAGGUACUCUCAUUGU 294 645-667 AD-85478 A-168469CCACCUUUUCUUCUAAUGAGU 111 2081-2101 A-170511 ACUCAUUAGAAGAAAAGGUGGGA 2952079-2101 AD-85479 A-168471 CGACCAGCUUGUUUGUGAAAA 112 2284-2304 A-170512UUUUCACAAACAAGCUGGUCGGU 296 2282-2304 AD-85480 A-168473ACCUUUUCUUCUAAUGAGUCA 113 2083-2103 A-170513 UGACUCAUUAGAAGAAAAGGUGG 2972081-2103 AD-85481 A-168475 GUCAUCCACAAUGAGAGUACA 114 638-658 A-170514UGUACUCUCAUUGUGGAUGACGA 298 636-658 AD-85482 A-168477CACAAUGAGAGUACCUGUGAA 115 644-664 A-170515 UUCACAGGUACUCUCAUUGUGGA 299642-664 AD-85483 A-168479 GUCUCCCACCUUUUCUUCUAA 116 2076-2096 A-170516UUAGAAGAAAAGGUGGGAGACUG 300 2074-2096 AD-85484 A-168481ACUUUCCAGCAAAACUCCCUA 117 1586-1606 A-170517 UAGGGAGUUUUGCUGGAAAGUGA 3011584-1606 AD-85485 A-168483 CCUCAACUGGAUGAAGAAACU 118 1603-1623 A-170518AGUUUCUUCAUCCAGUUGAGGGA 302 1601-1623 AD-85486 A-168485CUGUUUGCUGUGUAUGAUCAA 119 1889-1909 A-170519 UUGAUCAUACACAGCAAACAGGA 3031887-1909 AD-85487 A-168487 UUUGCUGUGUAUGAUCAAAGA 120 1892-1912 A-170520UCUUUGAUCAUACACAGCAAACA 304 1890-1912 AD-85488 A-168489CCGACCAGCUUGUUUGUGAAA 121 2283-2303 A-170521 UUUCACAAACAAGCUGGUCGGUU 3052281-2303 AD-85489 A-168491 UCCAACCGACCAGCUUGUUUA 122 2278-2298 A-170522UAAACAAGCUGGUCGGUUGGAAU 306 2276-2298 AD-85490 A-168493CCAUUCCUGUUUGCUGUGUAU 123 1883-1903 A-170523 AUACACAGCAAACAGGAAUGGGC 3071881-1903 AD-85491 A-168495 CACCUUUUCUUCUAAUGAGUA 124 2082-2102 A-170524UACUCAUUAGAAGAAAAGGUGGG 308 2080-2102 AD-85492 A-168497GUUUGCUGUGUAUGAUCAAAA 125 1891-1911 A-170525 UUUUGAUCAUACACAGCAAACAG 3091889-1911 AD-85493 A-168499 GCUGAGAAGAUUGACAGGUUA 126 1250-1270 A-170526UAACCUGUCAAUCUUCUCAGCAG 310 1248-1270 AD-85494 A-168501UUCCAGCAAAACUCCCUCAAA 127 1589-1609 A-170527 UUUGAGGGAGUUUUGCUGGAAAG 3111587-1609 AD-85495 A-168503 UGCUGAGAAGAUUGACAGGUU 128 1249-1269 A-170528AACCUGUCAAUCUUCUCAGCAGC 312 1247-1269 AD-85496 A-168505UCUCACUUUCCAGCAAAACUA 129 1582-1602 A-170529 UAGUUUUGCUGGAAAGUGAGACC 3131580-1602 AD-85497 A-168507 UCCACAAUGAGAGUACCUGUA 130 642-662 A-170530UACAGGUACUCUCAUUGUGGAUG 314 640-662 AD-85498 A-168509CCACCUCGUCAUCCACAAUGA 131 631-651 A-170531 UCAUUGUGGAUGACGAGGUGGAA 315629-651 AD-85499 A-168511 UCACUUUCCAGCAAAACUCCA 132 1584-1604 A-170532UGGAGUUUUGCUGGAAAGUGAGA 316 1582-1604 AD-85500 A-168513UCCCUCAACUGGAUGAAGAAA 133 1601-1621 A-170533 UUUCUUCAUCCAGUUGAGGGAGU 3171599-1621 AD-85501 A-168515 GAGAGUACCUGUGAGCAGCUA 134 650-670 A-170534UAGCUGCUCACAGGUACUCUCAU 318 648-670 AD-85502 A-168517AGAAUUCCAACCGACCAGCUU 135 2273-2293 A-170535 AAGCUGGUCGGUUGGAAUUCUUU 3192271-2293 AD-85503 A-168519 CAUUCCUGUUUGCUGUGUAUA 136 1884-1904 A-170536UAUACACAGCAAACAGGAAUGGG 320 1882-1904 AD-85504 A-168521GAAUUCCAACCGACCAGCUUA 137 2274-2294 A-170537 UAAGCUGGUCGGUUGGAAUUCUU 3212272-2294 AD-85505 A-168523 CAUCCACAAUGAGAGUACCUA 138 640-660 A-170538UAGGUACUCUCAUUGUGGAUGAC 322 638-660 AD-85506 A-168525CCCAUUCCUGUUUGCUGUGUA 139 1882-1902 A-170539 UACACAGCAAACAGGAAUGGGCG 3231880-1902 AD-85507 A-168527 CUGGGUUUAUUUUAGAGAAUA 140 2202-2222 A-170540UAUUCUCUAAAAUAAACCCAGCA 324 2200-2222 AD-85508 A-168529GCUGGGUUUAUUUUAGAGAAU 141 2201-2221 A-170541 AUUCUCUAAAAUAAACCCAGCAA 3252199-2221 AD-85509 A-168531 AUGGCAUGCACAGUGAGCUAU 142 861-881 A-170542AUAGCUCACUGUGCAUGCCAUAU 326 859-881 AD-85510 A-168533GAGAGAGCCCACAGAGUCUAA 143 1816-1836 A-170543 UUAGACUCUGUGGGCUCUCUCUC 3271814-1836 AD-85511 A-168535 GCAAGAACCAGUGUUUAGCGA 144 2234-2254 A-170544UCGCUAAACACUGGUUCUUGCCU 328 2232-2254 AD-85512 A-168537CCAGCAAAACUCCCUCAACUA 145 1591-1611 A-170545 UAGUUGAGGGAGUUUUGCUGGAA 3291589-1611 AD-85513 A-168539 CACCUCGUCAUCCACAAUGAA 146 632-652 A-170546UUCAUUGUGGAUGACGAGGUGGA 330 630-652 AD-85514 A-168541CGUCAUCCACAAUGAGAGUAA 147 637-657 A-170547 UUACUCUCAUUGUGGAUGACGAG 331635-657 AD-85515 A-168543 CUCCCACGCUCUCUGGACUUA 148 1211-1231 A-170548UAAGUCCAGAGAGCGUGGGAGGA 332 1209-1231 AD-85516 A-168545AACUCCCUCAACUGGAUGAAA 149 1598-1618 A-170549 UUUCAUCCAGUUGAGGGAGUUUU 3331596-1618 AD-85517 A-168547 UGAGAAGAUUGACAGGUUCAU 150 1252-1272 A-170550AUGAACCUGUCAAUCUUCUCAGC 334 1250-1272 AD-85518 A-168549CCUCGUCAUCCACAAUGAGAA 151 634-654 A-170551 UUCUCAUUGUGGAUGACGAGGUG 335632-654 AD-85519 A-168551 GAGAAGAUUGACAGGUUCAUA 152 1253-1273 A-170552UAUGAACCUGUCAAUCUUCUCAG 336 1251-1273 AD-85520 A-168553CUUCUUGGGCUUCCGUAUAUA 153 841-861 A-170553 UAUAUACGGAAGCCCAAGAAGUU 337839-861 AD-85521 A-168555 UCCACCUCGUCAUCCACAAUA 154 630-650 A-170554UAUUGUGGAUGACGAGGUGGAAG 338 628-650 AD-85522 A-168557AGAUUGACAGGUUCAUGCAGA 155 1257-1277 A-170555 UCUGCAUGAACCUGUCAAUCUUC 3391255-1277 AD-85523 A-168559 CUCCCUCAACUGGAUGAAGAA 156 1600-1620 A-170556UUCUUCAUCCAGUUGAGGGAGUU 340 1598-1620 AD-85524 A-168561AAUGAGAGUACCUGUGAGCAA 157 647-667 A-170557 UUGCUCACAGGUACUCUCAUUGU 341645-667 AD-85619 A-168469 CCACCUUUUCUUCUAAUGAGU 158 2081-2101 A-170558ACUCAUUAGAAGAAAAGGUGGGA 342 2079-2101 AD-85620 A-168471CGACCAGCUUGUUUGUGAAAA 159 2284-2304 A-170559 UUUUCACAAACAAGCUGGUCGGU 3432282-2304 AD-85621 A-168473 ACCUUUUCUUCUAAUGAGUCA 160 2083-2103 A-170560UGACUCAUUAGAAGAAAAGGUGG 344 2081-2103 AD-85622 A-168475GUCAUCCACAAUGAGAGUACA 161 638-658 A-170561 UGUACUCUCAUUGUGGAUGACGA 345636-658 AD-85623 A-168477 CACAAUGAGAGUACCUGUGAA 162 644-664 A-170562UUCACAGGUACUCUCAUUGUGGA 346 642-664 AD-85624 A-168479GUCUCCCACCUUUUCUUCUAA 163 2076-2096 A-170563 UUAGAAGAAAAGGUGGGAGACUG 3472074-2096 AD-85625 A-168481 ACUUUCCAGCAAAACUCCCUA 164 1586-1606 A-170564UAGGGAGUUUUGCUGGAAAGUGA 348 1584-1606 AD-85626 A-168483CCUCAACUGGAUGAAGAAACU 165 1603-1623 A-170565 AGUUUCUUCAUCCAGUUGAGGGA 3491601-1623 AD-85627 A-168485 CUGUUUGCUGUGUAUGAUCAA 166 1889-1909 A-170566UUGAUCAUACACAGCAAACAGGA 350 1887-1909 AD-85628 A-168487UUUGCUGUGUAUGAUCAAAGA 167 1892-1912 A-170567 UCUUUGAUCAUACACAGCAAACA 3511890-1912 AD-85629 A-168489 CCGACCAGCUUGUUUGUGAAA 168 2283-2303 A-170568UUUCACAAACAAGCUGGUCGGUU 352 2281-2303 AD-85630 A-168491UCCAACCGACCAGCUUGUUUA 169 2278-2298 A-170569 UAAACAAGCUGGUCGGUUGGAAU 3532276-2298 AD-85631 A-168493 CCAUUCCUGUUUGCUGUGUAU 170 1883-1903 A-170570AUACACAGCAAACAGGAAUGGGC 354 1881-1903 AD-85632 A-168495CACCUUUUCUUCUAAUGAGUA 171 2082-2102 A-170571 UACUCAUUAGAAGAAAAGGUGGG 3552080-2102 AD-85633 A-168497 GUUUGCUGUGUAUGAUCAAAA 172 1891-1911 A-170572UUUUGAUCAUACACAGCAAACAG 356 1889-1911 AD-85634 A-168499GCUGAGAAGAUUGACAGGUUA 173 1250-1270 A-170573 UAACCUGUCAAUCUUCUCAGCAG 3571248-1270 AD-85635 A-168501 UUCCAGCAAAACUCCCUCAAA 174 1589-1609 A-170574UUUGAGGGAGUUUUGCUGGAAAG 358 1587-1609 AD-85636 A-168503UGCUGAGAAGAUUGACAGGUU 175 1249-1269 A-170575 AACCUGUCAAUCUUCUCAGCAGC 3591247-1269 AD-85637 A-168505 UCUCACUUUCCAGCAAAACUA 176 1582-1602 A-170576UAGUUUUGCUGGAAAGUGAGACC 360 1580-1602 AD-85638 A-168507UCCACAAUGAGAGUACCUGUA 177 642-662 A-170577 UACAGGUACUCUCAUUGUGGAUG 361640-662 AD-85639 A-168509 CCACCUCGUCAUCCACAAUGA 178 631-651 A-170578UCAUUGUGGAUGACGAGGUGGAA 362 629-651 AD-85640 A-168511UCACUUUCCAGCAAAACUCCA 179 1584-1604 A-170579 UGGAGUUUUGCUGGAAAGUGAGA 3631582-1604 AD-85641 A-168513 UCCCUCAACUGGAUGAAGAAA 180 1601-1621 A-170580UUUCUUCAUCCAGUUGAGGGAGU 364 1599-1621 AD-85642 A-168515GAGAGUACCUGUGAGCAGCUA 181 650-670 A-170581 UAGCUGCUCACAGGUACUCUCAU 365648-670 AD-85643 A-168517 AGAAUUCCAACCGACCAGCUU 182 2273-2293 A-170582AAGCUGGUCGGUUGGAAUUCUUU 366 2271-2293 AD-85644 A-168519CAUUCCUGUUUGCUGUGUAUA 183 1884-1904 A-170583 UAUACACAGCAAACAGGAAUGGG 3671882-1904 AD-85645 A-168521 GAAUUCCAACCGACCAGCUUA 184 2274-2294 A-170584UAAGCUGGUCGGUUGGAAUUCUU 368 2272-2294 AD-85646 A-168523CAUCCACAAUGAGAGUACCUA 185 640-660 A-170585 UAGGUACUCUCAUUGUGGAUGAC 369638-660 AD-85647 A-168525 CCCAUUCCUGUUUGCUGUGUA 186 1882-1902 A-170586UACACAGCAAACAGGAAUGGGCG 370 1880-1902 AD-85648 A-168527CUGGGUUUAUUUUAGAGAAUA 187 2202-2222 A-170587 UAUUCUCUAAAAUAAACCCAGCA 3712200-2222 AD-85649 A-168529 GCUGGGUUUAUUUUAGAGAAU 188 2201-2221 A-170588AUUCUCUAAAAUAAACCCAGCAA 372 2199-2221 AD-85650 A-168531AUGGCAUGCACAGUGAGCUAU 189 861-881 A-170589 AUAGCUCACUGUGCAUGCCAUAU 373859-881 AD-85651 A-168533 GAGAGAGCCCACAGAGUCUAA 190 1816-1836 A-170590UUAGACUCUGUGGGCUCUCUCUC 374 1814-1836 AD-85652 A-168535GCAAGAACCAGUGUUUAGCGA 191 2234-2254 A-170591 UCGCUAAACACUGGUUCUUGCCU 3752232-2254 AD-85653 A-168537 CCAGCAAAACUCCCUCAACUA 192 1591-1611 A-170592UAGUUGAGGGAGUUUUGCUGGAA 376 1589-1611 AD-85654 A-168539CACCUCGUCAUCCACAAUGAA 193 632-652 A-170593 UUCAUUGUGGAUGACGAGGUGGA 377630-652 AD-85655 A-168541 CGUCAUCCACAAUGAGAGUAA 194 637-657 A-170594UUACUCUCAUUGUGGAUGACGAG 378 635-657 AD-85656 A-168543CUCCCACGCUCUCUGGACUUA 195 1211-1231 A-170595 UAAGUCCAGAGAGCGUGGGAGGA 3791209-1231 AD-85657 A-168545 AACUCCCUCAACUGGAUGAAA 196 1598-1618 A-170596UUUCAUCCAGUUGAGGGAGUUUU 380 1596-1618 AD-85658 A-168547UGAGAAGAUUGACAGGUUCAU 197 1252-1272 A-170597 AUGAACCUGUCAAUCUUCUCAGC 3811250-1272 AD-85659 A-168549 CCUCGUCAUCCACAAUGAGAA 198 634-654 A-170598UUCUCAUUGUGGAUGACGAGGUG 382 632-654 AD-85660 A-168551GAGAAGAUUGACAGGUUCAUA 199 1253-1273 A-170599 UAUGAACCUGUCAAUCUUCUCAG 3831251-1273 AD-85661 A-168553 CUUCUUGGGCUUCCGUAUAUA 200 841-861 A-170600UAUAUACGGAAGCCCAAGAAGUU 384 839-861 AD-85662 A-168555UCCACCUCGUCAUCCACAAUA 201 630-650 A-170601 UAUUGUGGAUGACGAGGUGGAAG 385628-650 AD-85663 A-168557 AGAUUGACAGGUUCAUGCAGA 202 1257-1277 A-170602UCUGCAUGAACCUGUCAAUCUUC 386 1255-1277 AD-85664 A-168559CUCCCUCAACUGGAUGAAGAA 203 1600-1620 A-170603 UUCUUCAUCCAGUUGAGGGAGUU 3871598-1620 AD-85665 A-168561 AAUGAGAGUACCUGUGAGCAA 204 647-667 A-170604UUGCUCACAGGUACUCUCAUUGU 388 645-667

TABLE 4 AGT Single 10 nM and 0.1 nM Dose Screen in Hep3B cells Duplex 10nM 10 nM 0.1 nM 0.1 nM ID Avg. SD Avg. SD AD-67327 6.2 1.5 30.1 5.9AD-84700 14.5 5.7 127.9 53.6 AD-84701 8.7 6.5 59.7 5.5 AD-84702 11.4 4.295.1 14.6 AD-84703 6.7 4.2 36.7 2.4 AD-84704 7.2 4.3 54.9 5.5 AD-847057.8 2.3 52.7 10.3 AD-84706 6.3 4.3 65.0 13.0 AD-84707 10.2 9.2 52.2 2.6AD-84708 10.1 4.1 98.7 24.6 AD-84709 19.0 4.3 102.7 18.5 AD-84710 11.37.9 65.9 19.0 AD-84711 10.4 6.5 66.5 13.8 AD-84712 7.5 6.4 66.4 13.9AD-84713 9.7 5.9 66.4 5.6 AD-84714 13.7 2.0 89.0 28.9 AD-84715 7.7 5.359.0 2.7 AD-84716 7.4 2.4 45.9 15.0 AD-84717 14.6 4.2 102.9 15.8AD-84718 6.8 0.7 66.9 8.3 AD-84719 21.1 4.0 92.5 19.6 AD-84720 11.3 0.496.0 22.1 AD-84721 14.7 5.2 88.4 12.7 AD-84722 35.9 14.8 106.6 21.2AD-84723 13.4 6.5 83.7 25.9 AD-84724 20.8 6.7 108.0 17.6 AD-84725 17.41.2 100.5 22.3 AD-84726 9.7 0.6 73.1 9.0 AD-84727 16.5 5.0 102.4 20.5AD-84728 17.6 2.6 103.6 12.8 AD-84729 11.6 3.4 78.9 16.1 AD-84730 11.21.5 77.6 8.7 AD-84731 11.4 2.9 80.4 15.9 AD-84732 26.9 2.6 88.2 19.5AD-84733 21.4 2.2 101.5 13.9 AD-84734 20.4 1.9 92.8 20.5 AD-84735 11.41.9 85.0 6.0 AD-84736 8.4 2.5 68.0 20.7 AD-84737 22.9 4.0 80.0 13.1AD-84738 16.0 1.8 98.3 29.5 AD-84739 6.6 2.1 40.5 6.8 AD-84740 10.2 1.878.9 32.7 AD-84741 6.3 1.0 37.0 9.7 AD-84742 14.1 2.0 86.9 7.4 AD-8474380.9 9.9 97.6 16.5 AD-84744 23.1 5.8 93.4 18.9 AD-84745 27.8 5.0 79.225.3 AD-84746 7.5 1.2 48.0 14.4 AD-85431 12.3 3.9 61.8 29.6 AD-8543210.1 6.6 50.5 19.8 AD-85433 11.5 4.0 61.4 25.4 AD-85434 5.8 2.5 37.220.4 AD-85435 7.3 1.8 44.9 17.8 AD-85436 8.2 4.9 35.2 12.8 AD-85437 7.26.3 30.4 18.6 AD-85438 10.4 7.5 41.8 18.1 AD-85439 15.4 3.8 62.1 25.9AD-85440 26.1 5.1 85.5 34.7 AD-85441 9.9 7.4 50.6 19.3 AD-85442 8.4 5.252.4 20.4 AD-85443 11.5 12.9 59.0 25.0 AD-85444 8.4 4.4 48.6 24.9AD-85445 21.7 6.8 63.2 36.1 AD-85446 14.7 11.5 59.0 27.5 AD-85447 8.42.9 52.8 15.5 AD-85448 12.6 4.0 89.5 25.2 AD-85449 8.3 1.5 72.7 14.2AD-85450 35.3 6.3 91.4 30.0 AD-85451 17.7 2.3 80.7 20.8 AD-85452 21.55.2 73.3 27.4 AD-85453 60.5 32.6 69.0 33.9 AD-85454 21.8 8.6 63.2 23.6AD-85455 34.0 12.4 88.8 23.4 AD-85456 21.6 1.4 91.9 18.1 AD-85457 12.12.6 85.6 28.0 AD-85458 29.8 7.1 98.1 25.4 AD-85459 23.8 2.0 101.2 34.7AD-85460 9.9 1.8 69.0 12.7 AD-85461 11.0 3.7 60.1 13.4 AD-85462 25.7 4.178.5 6.8 AD-85463 56.6 13.1 89.7 20.5 AD-85464 21.8 6.3 99.8 34.4AD-85465 25.2 6.2 99.2 24.6 AD-85466 35.3 8.4 104.2 31.5 AD-85467 12.72.7 82.2 8.4 AD-85468 30.9 6.5 93.0 31.2 AD-85469 44.7 8.0 82.1 13.4AD-85470 7.9 4.6 66.7 11.2 AD-85471 15.8 3.7 95.3 14.0 AD-85472 9.3 3.371.3 15.1 AD-85473 21.5 3.9 102.2 22.4 AD-85474 100.2 24.5 110.4 21.9AD-85475 28.7 8.6 105.2 31.1 AD-85476 35.5 10.2 81.4 13.1 AD-85477 10.12.2 76.9 30.6 AD-85478 15.1 1.9 94.6 27.5 AD-85479 18.7 5.8 92.1 26.6AD-85480 11.8 4.1 68.8 16.7 AD-85481 5.3 1.1 32.9 8.7 AD-85482 7.1 3.452.7 11.5 AD-85483 7.9 3.0 61.1 15.3 AD-85484 11.5 3.3 84.6 8.8 AD-8548511.6 5.5 58.9 19.7 AD-85486 25.3 6.2 94.8 21.4 AD-85487 40.5 10.2 101.024.4 AD-85488 32.8 7.4 97.6 24.2 AD-85489 12.8 4.0 87.2 20.9 AD-854907.6 0.8 78.5 20.2 AD-85491 12.5 4.5 92.3 19.0 AD-85492 89.7 17.7 112.320.2 AD-85493 6.3 2.6 51.0 14.5 AD-85494 11.0 2.4 79.8 24.0 AD-8549510.4 2.8 70.1 23.2 AD-85496 6.8 2.2 55.0 20.8 AD-85497 72.4 18.6 108.536.5 AD-85498 7.3 2.3 85.3 26.5 AD-85499 94.7 6.8 104.2 24.5 AD-8550063.1 21.9 98.4 21.4 AD-85501 38.3 12.3 129.0 47.3 AD-85502 27.2 8.3 92.021.9 AD-85503 36.2 11.5 108.3 25.4 AD-85504 9.2 3.7 56.1 9.4 AD-8550550.3 12.2 111.6 15.5 AD-85506 40.8 8.7 109.4 31.6 AD-85507 7.7 2.2 64.322.6 AD-85508 82.0 21.9 100.9 29.0 AD-85509 20.9 5.5 101.5 23.0 AD-85510104.0 28.6 90.1 42.0 AD-85511 14.1 2.6 84.2 9.3 AD-85512 50.6 14.7 98.530.7 AD-85513 35.0 9.0 78.5 24.4 AD-85514 13.0 0.9 80.3 27.9 AD-8551569.1 5.7 72.5 35.1 AD-85516 54.0 16.1 88.4 38.9 AD-85517 7.4 1.3 47.97.0 AD-85518 12.0 4.6 79.4 42.4 AD-85519 8.9 2.5 54.6 6.2 AD-85520 10.93.0 81.3 19.1 AD-85521 79.1 15.6 108.4 35.4 AD-85522 51.3 10.1 114.727.9 AD-85523 23.6 2.9 91.3 26.6 AD-85524 9.9 2.8 46.9 10.8 AD-8561920.7 1.0 114.4 53.5 AD-85620 20.0 5.8 96.8 25.7 AD-85621 8.9 6.9 80.420.3 AD-85622 6.4 3.7 50.2 12.9 AD-85623 7.6 6.0 38.0 4.6 AD-85624 9.23.7 81.0 15.9 AD-85625 6.0 2.4 58.9 8.7 AD-85626 7.6 6.3 43.8 3.9AD-85627 25.5 8.3 103.0 20.0 AD-85628 39.0 7.3 114.5 19.9 AD-85629 23.618.5 86.1 14.8 AD-85630 17.2 15.9 83.0 22.5 AD-85631 10.1 6.2 79.0 18.2AD-85632 10.2 4.3 67.5 10.8 AD-85633 42.1 19.9 95.1 4.5 AD-85634 7.5 2.954.6 11.7 AD-85635 8.2 2.6 53.5 24.0 AD-85636 11.4 2.2 96.0 23.7AD-85637 9.9 5.9 58.2 17.0 AD-85638 53.1 6.2 112 31.1 AD-85639 13.7 2.196.9 19.6 AD-85640 72.8 11.7 106.9 7.2 AD-85641 47.2 19.8 89.6 9.5AD-85642 15.3 3.5 86.7 24.5 AD-85643 35.4 12.9 494.9 779.7 AD-85644 79.07.2 110.6 23.5 AD-85645 7.8 1.2 63.6 13.1 AD-85646 33.6 8.6 105.6 28.1AD-85647 19.4 3.1 102.3 9.7 AD-85648 49.0 6.6 106.0 10.7 AD-85649 18.52.9 82.4 10.1 AD-85650 69.4 19.0 87.2 15.6 AD-85651 90.4 17.3 106.9 31.9AD-85652 31.3 8.5 90.0 37.7 AD-85653 47.6 2.8 104.1 20.6 AD-85654 66.73.7 97.5 17.8 AD-85655 10.4 0.8 55.4 6.9 AD-85656 96.7 4.3 98.6 23.4AD-85657 83.6 3.3 86.6 31.4 AD-85658 7.6 1.8 65.4 9.2 AD-85659 14.1 4.276.0 33.0 AD-85660 40.9 6.5 95.8 24.0 AD-85661 63.6 10.4 93.0 34.1AD-85662 83.4 11.5 87.3 17.2 AD-85663 23.0 5.3 79.1 30.8 AD-85664 44.82.9 112.2 19.9 AD-85665 13.5 2.5 81.0 25.4

TABLE 5Modified Sense and Antisense Strand Sequences of hAGT dsRNA AgentsDuplex Name Modified Sense Sequence 5′ to 3′ SEQ ID NO:Modified Antisense Sequence 5′ to 3′ SEQ ID NO:mRNA target sequence 5′ to 3′ SEQ ID NO: AD-84704csascaauGfaGfAfGfuaccugugaaL96 389 usUfscacAfgGfUfacucUfcAfuugugsgsa 573UCCACAAUGAGAGUACCUGUGAG 757 AD-84705 gsuscuccCfaCfCfUfuuucuucuaaL96 390usUfsagaAfgAfAfaaggUfgGfgagacsusg 574 CAGUCUCCCACCUUUUCUUCUAA 758AD-84706 ascsuuucCfaGfCfAfaaacucccuaL96 391usAfsgggAfgUfUfuugcUfgGfaaagusgsa 575 UCACUUUCCAGCAAAACUCCCUC 759AD-84707 cscsucaaCfuGfGfAfugaagaaacuL96 392asGfsuuuCfuUfCfauccAfgUfugaggsgsa 576 UCCCUCAACUGGAUGAAGAAACU 760AD-84708 csusguuuGfcUfGfUfguaugaucaaL96 393usUfsgauCfaUfAfcacaGfcAfaacagsgsa 577 UCCUGUUUGCUGUGUAUGAUCAA 761AD-84709 ususugcuGfuGfUfAfugaucaaagaL96 394usCfsuuuGfaUfCfauacAfcAfgcaaascsa 578 UGUUUGCUGUGUAUGAUCAAAGC 762AD-84710 cscsgaccAfgCfUfUfguuugugaaaL96 395usUfsucaCfaAfAfcaagCfuGfgucggsusu 579 AACCGACCAGCUUGUUUGUGAAA 763AD-84711 uscscaacCfgAfCfCfagcuuguuuaL96 396usAfsaacAfaGfCfugguCfgGfuuggasasu 580 AUUCCAACCGACCAGCUUGUUUG 764AD-84712 cscsauucCfuGfUfUfugcuguguauL96 397asUfsacaCfaGfCfaaacAfgGfaauggsgsc 581 GCCCAUUCCUGUUUGCUGUGUAU 765AD-84713 csasccuuUfuCfUfUfcuaaugaguaL96 398usAfscucAfuUfAfgaagAfaAfaggugsgsg 582 CCCACCUUUUCUUCUAAUGAGUC 766AD-84714 gsusuugcUfgUfGfUfaugaucaaaaL96 399usUfsuugAfuCfAfuacaCfaGfcaaacsasg 583 CUGUUUGCUGUGUAUGAUCAAAG 767AD-84715 gscsugagAfaGfAfUfugacagguuaL96 400usAfsaccUfgUfCfaaucUfuCfucagcsasg 584 CUGCUGAGAAGAUUGACAGGUUC 768AD-84716 ususccagCfaAfAfAfcucccucaaaL96 401usUfsugaGfgGfAfguuuUfgCfuggaasasg 585 CUUUCCAGCAAAACUCCCUCAAC 769AD-84717 usgscugaGfaAfGfAfuugacagguuL96 402asAfsccuGfuCfAfaucuUfcUfcagcasgsc 586 GCUGCUGAGAAGAUUGACAGGUU 770AD-84718 uscsucacUfuUfCfCfagcaaaacuaL96 403usAfsguuUfuGfCfuggaAfaGfugagascsc 587 GGUCUCACUUUCCAGCAAAACUC 771AD-84719 uscscacaAfuGfAfGfaguaccuguaL96 404usAfscagGfuAfCfucucAfuUfguggasusg 588 CAUCCACAAUGAGAGUACCUGUG 772AD-84720 cscsaccuCfgUfCfAfuccacaaugaL96 405usCfsauuGfuGfGfaugaCfgAfgguggsasa 589 UUCCACCUCGUCAUCCACAAUGA 773AD-84721 uscsacuuUfcCfAfGfcaaaacuccaL96 406usGfsgagUfuUfUfgcugGfaAfagugasgsa 590 UCUCACUUUCCAGCAAAACUCCC 774AD-84722 uscsccucAfaCfUfGfgaugaagaaaL96 407usUfsucuUfcAfUfccagUfuGfagggasgsu 591 ACUCCCUCAACUGGAUGAAGAAA 775AD-84723 gsasgaguAfcCfUfGfugagcagcuaL96 408usAfsgcuGfcUfCfacagGfuAfcucucsasu 592 AUGAGAGUACCUGUGAGCAGCUG 776AD-84724 asgsaauuCfcAfAfCfcgaccagcuuL96 409asAfsgcuGfgUfCfgguuGfgAfauucususu 593 AAAGAAUUCCAACCGACCAGCUU 777AD-84725 csasuuccUfgUfUfUfgcuguguauaL96 410usAfsuacAfcAfGfcaaaCfaGfgaaugsgsg 594 CCCAUUCCUGUUUGCUGUGUAUG 778AD-84726 gsasauucCfaAfCfCfgaccagcuuaL96 411usAfsagcUfgGfUfcgguUfgGfaauucsusu 595 AAGAAUUCCAACCGACCAGCUUG 779AD-84727 csasuccaCfaAfUfGfagaguaccuaL96 412usAfsgguAfcUfCfucauUfgUfggaugsasc 596 GUCAUCCACAAUGAGAGUACCUG 780AD-84728 cscscauuCfcUfGfUfuugcuguguaL96 413usAfscacAfgCfAfaacaGfgAfaugggscsg 597 CGCCCAUUCCUGUUUGCUGUGUA 781AD-84729 csusggguUfuAfUfUfuuagagaauaL96 414usAfsuucUfcUfAfaaauAfaAfcccagscsa 598 UGCUGGGUUUAUUUUAGAGAAUG 782AD-84730 gscsugggUfuUfAfUfuuuagagaauL96 415asUfsucuCfuAfAfaauaAfaCfccagcsasa 599 UUGCUGGGUUUAUUUUAGAGAAU 783AD-84731 asusggcaUfgCfAfCfagugagcuauL96 416asUfsagcUfcAfCfugugCfaUfgccausasu 600 AUAUGGCAUGCACAGUGAGCUAU 784AD-84732 gsasgagaGfcCfCfAfcagagucuaaL96 417usUfsagaCfuCfUfguggGfcUfcucucsusc 601 GAGAGAGAGCCCACAGAGUCUAC 785AD-84733 gscsaagaAfcCfAfGfuguuuagcgaL96 418usCfsgcuAfaAfCfacugGfuUfcuugcscsu 602 AGGCAAGAACCAGUGUUUAGCGC 786AD-84734 cscsagcaAfaAfCfUfcccucaacuaL96 419usAfsguuGfaGfGfgaguUfuUfgcuggsasa 603 UUCCAGCAAAACUCCCUCAACUG 787AD-84735 csasccucGfuCfAfUfccacaaugaaL96 420usUfscauUfgUfGfgaugAfcGfaggugsgsa 604 UCCACCUCGUCAUCCACAAUGAG 788AD-84736 csgsucauCfcAfCfAfaugagaguaaL96 421usUfsacuCfuCfAfuuguGfgAfugacgsasg 605 CUCGUCAUCCACAAUGAGAGUAC 789AD-84737 csuscccaCfgCfUfCfucuggacuuaL96 422usAfsaguCfcAfGfagagCfgUfgggagsgsa 606 UCCUCCCACGCUCUCUGGACUUC 790AD-84738 asascuccCfuCfAfAfcuggaugaaaL96 423usUfsucaUfcCfAfguugAfgGfgaguususu 607 AAAACUCCCUCAACUGGAUGAAG 791AD-84739 usgsagaaGfaUfUfGfacagguucauL96 424asUfsgaaCfcUfGfucaaUfcUfucucasgsc 608 GCUGAGAAGAUUGACAGGUUCAU 792AD-84740 cscsucguCfaUfCfCfacaaugagaaL96 425usUfscucAfuUfGfuggaUfgAfcgaggsusg 609 CACCUCGUCAUCCACAAUGAGAG 793AD-84741 gsasgaagAfuUfGfAfcagguucauaL96 426usAfsugaAfcCfUfgucaAfuCfuucucsasg 610 CUGAGAAGAUUGACAGGUUCAUG 794AD-84742 csusucuuGfgGfCfUfuccguauauaL96 427usAfsuauAfcGfGfaagcCfcAfagaagsusu 611 AACUUCUUGGGCUUCCGUAUAUA 795AD-84743 uscscaccUfcGfUfCfauccacaauaL96 428usAfsuugUfgGfAfugacGfaGfguggasasg 612 CUUCCACCUCGUCAUCCACAAUG 796AD-84744 asgsauugAfcAfGfGfuucaugcagaL96 429usCfsugcAfuGfAfaccuGfuCfaaucususc 613 GAAGAUUGACAGGUUCAUGCAGG 797AD-84745 csuscccuCfaAfCfUfggaugaagaaL96 430usUfscuuCfaUfCfcaguUfgAfgggagsusu 614 AACUCCCUCAACUGGAUGAAGAA 798AD-84746 asasugagAfgUfAfCfcugugagcaaL96 431usUfsgcuCfaCfAfgguaCfuCfucauusgsu 615 ACAAUGAGAGUACCUGUGAGCAG 799AD-85431 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AAAGAAUUCCAACCGACCAGCUU 918AD-85644 csasuuccUfgUfUfUfgcuguguauaL96 551usAfsuaca(Cgn)agcaaaCfaGfgaaugsgsg 735 CCCAUUCCUGUUUGCUGUGUAUG 919AD-85645 gsasauucCfaAfCfCfgaccagcuuaL96 552usAfsagcu(Ggn)gucgguUfgGfaauucsusu 736 AAGAAUUCCAACCGACCAGCUUG 920AD-85646 csasuccaCfaAfUfGfagaguaccuaL96 553usAfsggua(Cgn)ucucauUfgUfggaugsasc 737 GUCAUCCACAAUGAGAGUACCUG 921AD-85647 cscscauuCfcUfGfUfuugcuguguaL96 554usAfscaca(Ggn)caaacaGfgAfaugggscsg 738 CGCCCAUUCCUGUUUGCUGUGUA 922AD-85648 csusggguUfuAfUfUfuuagagaauaL96 555usAfsuucu(Cgn)uaaaauAfaAfcccagscsa 739 UGCUGGGUUUAUUUUAGAGAAUG 923AD-85649 gscsugggUfuUfAfUfuuuagagaauL96 556asUfsucuc(Tgn)aaaauaAfaCfccagcsasa 740 UUGCUGGGUUUAUUUUAGAGAAU 924AD-85650 asusggcaUfgCfAfCfagugagcuauL96 557asUfsagcu(Cgn)acugugCfaUfgccausasu 741 AUAUGGCAUGCACAGUGAGCUAU 925AD-85651 gsasgagaGfcCfCfAfcagagucuaaL96 558usUfsagac(Tgn)cuguggGfcUfcucucsusc 742 GAGAGAGAGCCCACAGAGUCUAC 926AD-85652 gscsaagaAfcCfAfGfuguuuagcgaL96 559usCfsgcua(Agn)acacugGfuUfcuugcscsu 743 AGGCAAGAACCAGUGUUUAGCGC 927AD-85653 cscsagcaAfaAfCfUfcccucaacuaL96 560usAfsguug(Agn)gggaguUfuUfgcuggsasa 744 UUCCAGCAAAACUCCCUCAACUG 928AD-85654 csasccucGfuCfAfUfccacaaugaaL96 561usUfscauu(Ggn)uggaugAfcGfaggugsgsa 745 UCCACCUCGUCAUCCACAAUGAG 929AD-85655 csgsucauCfcAfCfAfaugagaguaaL96 562usUfsacuc(Tgn)cauuguGfgAfugacgsasg 746 CUCGUCAUCCACAAUGAGAGUAC 930AD-85656 csuscccaCfgCfUfCfucuggacuuaL96 563usAfsaguc(Cgn)agagagCfgUfgggagsgsa 747 UCCUCCCACGCUCUCUGGACUUC 931AD-85657 asascuccCfuCfAfAfcuggaugaaaL96 564usUfsucau(Cgn)caguugAfgGfgaguususu 748 AAAACUCCCUCAACUGGAUGAAG 932AD-85658 usgsagaaGfaUfUfGfacagguucauL96 565asUfsgaac(Cgn)ugucaaUfcUfucucasgsc 749 GCUGAGAAGAUUGACAGGUUCAU 933AD-85659 cscsucguCfaUfCfCfacaaugagaaL96 566usUfscuca(Tgn)uguggaUfgAfcgaggsusg 750 CACCUCGUCAUCCACAAUGAGAG 934AD-85660 gsasgaagAfuUfGfAfcagguucauaL96 567usAfsugaa(Cgn)cugucaAfuCfuucucsasg 751 CUGAGAAGAUUGACAGGUUCAUG 935AD-85661 csusucuuGfgGfCfUfuccguauauaL96 568usAfsuaua(Cgn)ggaagcCfcAfagaagsusu 752 AACUUCUUGGGCUUCCGUAUAUA 936AD-85662 uscscaccUfcGfUfCfauccacaauaL96 569usAfsuugu(Ggn)gaugacGfaGfguggasasg 753 CUUCCACCUCGUCAUCCACAAUG 937AD-85663 asgsauugAfcAfGfGfuucaugcagaL96 570usCfsugca(Tgn)gaaccuGfuCfaaucususc 754 GAAGAUUGACAGGUUCAUGCAGG 938AD-85664 csuscccuCfaAfCfUfggaugaagaaL96 571usUfscuuc(Agn)uccaguUfgAfgggagsusu 755 AACUCCCUCAACUGGAUGAAGAA 939AD-85665 asasugagAfgUfAfCfcugugagcaaL96 572usUfsgcuc(Agn)cagguaCfuCfucauusgsu 756 ACAAUGAGAGUACCUGUGAGCAG 940

TABLE 6Additional Modified Sense and Antisense Strand Sequences of hAGT dsRNA AgentsSense SEQ Antisense Modified SEQ Duplex Oligo Modified Sense ID Range inOligo Antisense ID Range in Name Name Sequence 5′ to 3′ NO: NM_000029.3Name Sequence NO: NM_000029.3 AD-126306 A-168475 gsuscaucCfaCfAfAf 941638-658 A-250785 usGfsua(Cgn)ucuca 951 636-658 ugagaguacaL96uugUfgGfaugacsgsa AD-126307 A-168477 csascaauGfaGfAfGf 942 644-664A-250786 usUfsca(Cgn)aggua 952 642-664 uaccugugaaL96 cucUfcAfuugugsgsaAD-126308 A-168479 gsuscuccCfaCfCfUf 943 2076-2096 A-250787usUfsag(Agn)agaaa 953 2074-2096 uuucuucuaaL96 aggUfgGfgagacsusgAD-126310 A-168483 cscsucaaCfuGfGfAf 944 1603-1623 A-250789asGfsuu(Tgn)cuuca 954 1601-1623 ugaagaaacuL96 uccAfgUfugaggsgsaAD-126343 A-168549 cscsucguCfaUfCfCf 945 634-654 A-250822usUfscu(Cgn)auugu 955 632-654 acaaugagaaL96 ggaUfgAfcgaggsusg AD-133360A-168475 gsuscaucCfaCfAfAf 946 638-658 A-264752 usGfs(Tgn)acucuca 956636-658 ugagaguacaL96 uugUfgGfaugacsgsa AD-133361 A-168475gsuscaucCfaCfAfAf 947 638-658 A-264753 usGfsu(Agn)cucuca 957 636-658ugagaguacaL96 uugUfgGfaugacsgsa AD-133362 A-168475 gsuscaucCfaCfAfAf 948638-658 A-264754 usGfsuacuc(Tgn)ca 958 636-658 ugagaguacaL96uugUfgGfaugacsgsa AD-133374 A-168479 gsuscuccCfaCfCfUf 949 2076-2096A-264766 usUfsagaag(Agn)aa 959 2074-2096 uuucuucuaaL96 aggUfgGfgagacsusgAD-133385 A-168477 csascaauGfaGfAfGf 950 644-664 A-264777usUfsc(Agn)caggua 960 642-664 uaccugugaaL96 cucUfcAfuugugsgsa

Example 3. In Vivo Screening of dsRNA Duplexes in Mice Trandsuced withAAV Expressing Human AGT

To express human angiotensinogen, C57/BL6 mice were first transducedwith an AAV (adeno-associated virus) vector expressing the human AGTtranscript. After at least two weeks from AAV introduction, blood wasobtained from mice for baseline circulating human AGT levels, andanimals then received a single 3 mg/kg subcutaneous dose of one of asubset of the dsRNA agents provided in Tables 5 and 6 (N=3 per group).Blood was obtained from animals again at fourteen days post-dose ofdsRNA agent. Human AGT levels were quantified using an ELISA specificfor human angiotensinogen, according to manufacturer's protocol (IBLAmerica #27412). Data were expressed as percent of baseline value, andpresented as mean plus standard deviation. Certain dsRNA duplexes wereselected for further analysis.

TABLE 7 AGT Single 3 mg/kg Dose Screen in AAV-human AGT transduced mouseDuplexID Avg SD AD-67327 13.2 4.5 AD-85110 97.0 3.6 AD-85117 86.7 10.8AD-85118 107.2 14.2 AD-85434 18.4 0.6 AD-85435 25.7 1.7 AD-85438 12.62.7 AD-85446 8.4 1.0 AD-85481 27.0 11.5 AD-85482 64.2 13.0 AD-85482 48.55.9 AD-85483 38.9 4.6 AD-85485 63.6 3.0 AD-85493 35.7 20.5 AD-85496 78.510.3 AD-85517 93.8 4.6 AD-85524 90.4 14.7 AD-85622 38.1 10.6 AD-8562328.0 8.4 AD-85625 87.6 13.7 AD-85626 29.9 14.0 AD-85634 27.7 4.5AD-85635 87.1 13.5 AD-126306 12.4 1.4 AD-126307 21.6 9.0 AD-126308 15.71.8 AD-126310 38.3 1.1 AD-126343 65.5 13.4 AD-133360 50.6 7.5 AD-13336123.2 12.4 AD-133362 12.2 4.1 AD-133374 26.2 3.3 AD-133385 33.8 8.7

Example 4. In Vivo Screening of dsRNA Duplexes in Cynomolgus Monkeys

Duplexes of interest, identified from the above mouse studies, wereevaluated in cynomolgus monkey. Animals (N=3 per group) received asingle 3 mg/kg subcutaneous dose of a dsRNA agent (AD-85481, AD-126306,AD-126307, AD-126308, or AD-133362) on day 1. Blood was obtained on days−6, 1, 4, 8, 15, 22, 29, 32, 35, 43, 57, 71, 85, and 99 post-dose.Circulating AGT levels were quantified using an ELISA specific for humanangiotensinogen (and cross-reactive with cynomolgus), according tomanufacturer's protocol (IBL America #27412). Data were expressed aspercent of baseline value, and presented as mean plus/minus standarddeviation. The results are showin in FIG. 1A. The apparent differencesbetween AD-85481, AD-126306, and AD-133362 are within the range oftypical study-to-study variability in non-human primates.

Dose-response studies were performed to assess the activity of AD-67327and AD-85481. Although the experiments were performed separately, themethods used were essentially the same and the results are presentedtogether in FIG. 1B. Cynomolgus monkeys received a single 0.3 mg/kg, 1mg/kg, or 3 mg/kg subcutaneous dose of dsRNA agent on day 1. Blood wasobtained on days −6, 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71 and 78post-dose for AD-67327 and on days −6, 1, 4, 8, 15, 22, 29, 32, 35, 43,57, 71, 85, and 99 post-dose for AD-85481. Circulating AGT levels werequantified using an ELISA specific for human angiotensinogen (andcross-reactive with cynomolgus), according to manufacturer's protocol(IBL America #27412). Data were expressed as percent of baseline value,and presented as mean plus standard deviation. The results are shown inFIG. 1B. These data demonstrate a roughly 3-fold improvement in efficacyand duration for AD-85481 over AD-67327.

Multidose studies were performed to determine the potency and durabilityof AD-67327 and AD-85481. Although the experiments were performedseparately, the methods used were essentially the same and the resultsare presented together in FIG. 1C. Cynomolgus monkeys weresubcutaneously administered a 1 mg/kg dose of AD-67327 or AD-85481 onceevery four weeks for three weeks (q4w dosing) (days 1, 29, and 57 postfirst dose). Blood was obtained for evaluation of circulating AGT atdays −6, 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71 and 78 post first doseof AD-67327 and at days −8, 1, 4, 8, 15, 22, 29, 36, 43, 57, 64, 71, 85,99 post first dose of AD-85481. These data demonstrate an increase inpotency and durability of target silencing by AD-85481 compared toAD-67327.

Example 5. Treatment of Hypertension with an AGT dsRNA in a SpontaneousHypertensive Rat Model

A rat specific dsRNA was designed to test the effect of AGT knockdown ina spontaneously hypertensive rat model. Spontaneously hypertensive rats(N=9 per group) were subcutaneously administered a 10 mg/kg dose of therat-specific AGT dsRNA once every 2 weeks (10 mg/kg q2w), or daily oraldoses of the ARB valsartan (31 mg/kg/day), or daily oral doses of theACE inhibitor captopril (100 mg/kg/day). Select combinations (the ratspecific dsRNA agent plus valsartan or captopril plus valsartan) werealso evaluated, dosed as noted above. Mean arterial pressure wasmeasured by telemetry over a 4-week period. After four weeks oftreatment, animals were anaesthetized by i.p. pentobarbital injection,and blood collected from the hepatic portal vein for the measurement ofplasma AGT, plasma Renin, plasma Angiotensin II, plasma aldosterone,plasma K+, and plasma Renin activity. Heart weights and tibia length(for normalization of heart weights) were also obtained.

Treatment with the rat-specific dsRNA agent knocked down plasma AGTlevels by over 98% when administered alone or in combination withvalsartan relative to pre-treatment levels (FIG. 2A). Treatment with thecombination of valsartan and captopril was also demonstrated tosignificantly decrease serum AGT levels. All treatments increased thelevel of Renin, with the greatest increase occurring in animals treatedwith a combination of valsartan and the dsRNA agent, followed by asubstantial increase in the dsRNA agent alone-treated group. Onlycombination treatment with valsartan and the dsRNA agent was found tolower circulating Angiotensin II levels. A trend towards reduced urinaryAGT was observed after treatment with the dsRNA agent, suggesting thatlevels of AGT protein in the kidney are not significantly inhibited bytreatment with the dsRNA agent (FIG. 3). Only the combination ofvalsartan and the dsRNA agent was found to significantly lower urinaryAGT. No treatments altered aldosterone levels. Plasma K⁺ tended toincrease in all groups, with significance only being reached in thecombination valsartan plus siRNA treatment group.

FIG. 2B shows mean arterial pressure levels measured by telemetrythroughout the experiment and graphed relative to starting bloodpressure levels. Each of the treatments caused a statisticallysignificant decrease in blood pressure as compared to untreated animals.Statistical comparisons (p<0.05) are noted relative to baseline (#) orvalsartan plus captopril ($). Treatment with valsaratan plus therat-specific dsRNA agent was significantly better than treatment withcaptopril plus valsartan in lowering mean arterial pressure.

FIG. 2C shows heart weights normalized to tibial lengths to provide ameasure of cardiac hypertrophy. Treatment with both valsartan pluscaptopril and valsartan plus the dsRNA agent were effective at reducingcardiac hypertrophy relative to control (p<0.05), with valsartan plusthe dsRNA agent also reducing cardiac hypertrophy relative to valsartanplus captopril (p<0.05). FIG. 2E depicts the same data as a scatterplotof heart weight to tibial length versus MAP. A linear relationshipbetween cardiac hypertrophy and MAP is observed, with valsartan plus thedsRNA agent providing the greatest reduction in cardiac hypertrophy.Cardiomyocyte size was reduced relative to vehicle by all groups exceptvalsartan (FIG. 2F), while NT-proBNP was reduced in the captopril plusvalsartan group and a trend to reduction was observed in the valsartanplus dsRNA group (FIG. 2G).

FIG. 2D shows relative Renin activity at level at 4 weeks relative tobaseline. Plasma Renin activity (PRA), which reflects reducedangiotensin signaling, indicates a clear increase in PRA with the dsRNAagent treatment (p<0.05 relative to both baseline and control group, forboth the dsRNA agent alone and valsartan plus siRNA). The PRA assaymeasures Renin activity by quantifying the amount of Angiotensin Iproduced by Renin in a blood sample, in the presence of excessangiotensinogen. These data demonstrate a reduction in Angiotensin IIsignaling following that treatment with AGT-dsRNA agent, and that theeffect is enhanced by co-treatment with valsartan. Due to upregulation,circulating angiotensin II remains intact even when AGT levels arealmost completely knocked down. These data demonstrate that AGT-dsRNAagent causes a similar antihypertensive effect as valsartan andcaptopril. Without being bound by theory, it is proposed that only whencombining dsRNA agent plus valsartan do angiotensin II levels collapse,resulting in a synergistic decrease in blood pressure.

The effect of various treatments on blood and renal AngI and AngIIlevels was investigated after four weeks of treatment. FIGS. 4A-4C showthat treatment with valsartan and captopril, either alone or incombination, significantly increased blood levels of AngI as compared tovehicle control (FIG. 4A). The dsRNA agent alone did not significantlyalter blood levels of AngI, but the combination of valsartan with thedsRNA agent significantly decreased blood AngI as compared to vehiclecontrol and treatment with the dsRNA agent alone. Valsartan alone wasfound to significantly increase blood AngII as compared to vehiclecontrol (FIG. 4B). Combination of valsartan with the dsRNA agent wasfound to significantly decrease blood AngII as compared to vehiclecontrol. These changes resulted in a significant decrease in the ratioof AngII/AngI in the captopril and captopril+valsartan treated animals.The data for captopril and valsartan and consistent with theirmechanisms of action, while the data for the dsRNA alone indicateslittle effect on AngIII in the blood.

FIGS. 5A-5C show that the dsRNA agent reduced renal AngI withoutapparent effect on renal AngII, resulting in an upregulated renal AngIIIratio. FIG. 5A shows that each valsartan and captopril significantlyincreased renal AngI, while the combination of the agents did not have asignificant effect on the level of AngI. Renal AngI was significantlydecreased by the dsRNA agent and the combination of the dsRNA agent withvalsartan. Moreover, the combination treatment significantly reducedrenal AngI level as compared to treatment with the dsRNA agent alone.FIG. 5B shows no significant change in renal AngII after treatment withany of the monotherapies except captoril, i.e., valsartan or the dsRNAagent alone. However, the combination of captopril and valsartan and thecombination of valsartan and the dsRNA agent were demonstrated tosignificantly reduce renal Ang II. The data for captopril and valsartanare consistent with their mechanisms of action, while the data for thedsRNA alone indicates little apparent effect on AngII in the kidneys.The renal AngII/AngI ratio increased by 4-fold after administration ofthe AGT dsRNA agent alone (FIG. 5C). Conversely, a decrease of over 70%in the Ang II/Ang I ratio was seen after treatment with valsartan,captopril, and the combination of valsartan+captopril. No significantchange in the ratio of AngII/AngI was observed after treatment with thevalsartan+AGT dsRNA agent. The increase in renal AngII was demonstratedto not be a result of alterations in renal angiotensin receptor levelsor ACE mRNA expression in renal cortex or medulla. FIGS. 6A-6C show nosignificant changes in ATla receptor, ATIb receptor, or ACE mRNA levelin kidney under any treatment conditions except one. Treatment withcaptopril caused a significant increase in ATIb receptor level in kidneymedulla. The effect of the treatment regimens on kidney function wasalso assessed. No changes in glomerular filtration rate (GFR),natriuresis, and albuminuria were observed. This indicates that thesetreatments did not impair kidney function.

Urinary volume and urinary sodium were monitored during the experiment.Treatment with valsartan+dsRNA agent, valsartan+captopril, and captoprilalone caused a significant increase in urinary volume within groupsbetween baseline and 4 weeks (FIG. 7). Comparison of urinary volumeacross groups at 4 weeks showed a significant increase in urinary outputin captopril treated animals as compared to all other groups. Treatmentwith valsartan+dsRNA agent resulted in a significant increase in urinaryoutput at 4 weeks as compared to treatment with valsartan alone orvehicle. No significant changes in urinary sodium were observed acrossor within groups during the experiment.

These data demonstrate that the reduced renal Ang III ratio during bothACEi and ARB confirms that renal ACE generates Ang II, and that tissueAng II represents ATR-internalized Ang II (van Esch et al., CardiovascRes 2010 86(3):401-409). Further, the lowering of renal Ang I afterliver-targeted AGT siRNA treatment demonstrates that renal Anggeneration depends on hepatic AGT. Although, urinary AGT is partlykidney-derived, this renal AGT does not contribute to renal Anggeneration, as has been suggested before (Matsusaka et al., JASN 201223: 1181-1189). The increased renal Ang III ratio after AGT dsRNAtreatment, allowing renal Ang II levels to remain intact, is suggestivefor enhanced Ang II internalization, albeit in the absence of AT1breceptor upregulation. In agreement with this concept, additive ARBexposure virtually abolished renal Ang II.

Treatment with the liver-specific AGT dsRNA agent synergistically lowersarterial pressure when combined with existing RAS blockers and lowersrenal Ang production, without apparent negative effects on renalfunction.

Example 6. Treatment of Hypertension with an AGT dsRNA in a High SaltRat Model

The deoxycorticosterone acetate (DOCA)-salt rat model is a wellestablished model for hypertension in the context of high salt levels,and is considered a model of neurogenic hypertension due to the effecton central and peripheral nervous systems (Basting T & Lazartigues E,Cur Hypertension Rep 2017).

Upon arrival, Sprague-Dawley rats are allowed to acclimatize for 7 days.Subsequently, telemetry transmitters are implanted intra-abdominally, inthe abdominal aorta, in the rats under isoflurane anaesthesia. The ratsare allowed to recover from this procedure for 10 days. From thenonwards, blood pressure, heart rate, and other indicators ofhypertension are measured by telemetry over a 7-week time period. Duringthe first 4 weeks, animals are subcutaneoulsy implanted with a 200 mgDOCA pellet and receive 0.9% salt in the drinking water (ad libidum) ona chronic basis to induce hypertension. After this period during whichhypertension begins, a 3-week treatment period is initiated. Rats aretreated with

1) vehicle;

2) valsartan, 31 mg/kg/day added to drinking water;

3) an AGT dsRNA agent, 10 mg/kg once every two weeks, subcutaneously;

4) spironolactone, 50 mg/kg/day, subcutaneously; a combination of an AGTdsRNA agent, 10 mg/kg once every two weeks, subcutaneously, andvalsartan, 31 mg/kg/day added to drinking water;

5) an AGT dsRNA agent, 10 mg/kg once every two weeks, subcutaneously,and spironolactone, 50 mg/kg/day, subcutaneously;

6) valsartan, 31 mg/kg/day added to drinking water, and spironolactone,50 mg/kg/day, subcutaneously; or

7) an AGT dsRNA agent, 30 mg/kg once every two weeks, subcutaneously. Inaddition, one group of rats that do not receive DOCA and salt in thedrinking water serves as controls to evaluate the effect of thetreatments to normalize blood pressure.

Example 7. Treatment of Obesity with an AGT dsRNA Agent in a High FatFed Mouse Model of Diet Induced Obesity (DIO)

Sixteen-week old high fat fed (HFF) obese mice (diet-induced obesity(DIO)) and normal-weight control animals were purchased and kept ontheir respective high fat diet (60% of calories as fat) or normal chow.After acclimatization, animals were divided into four groups: Normalweight+PBS; Normal weight+AGT dsRNA; DIO+PBS; and DIO+AGT dsRNA(n=5/group). Animals received 10 mg/kg mouse-specific dsRNA or PBS everyother week for 12 weeks starting at week 0. Animals were weighed andblood obtained biweekly. Serum AGT levels were determined by ELISA. Afasting glucose tolerance test was performed predose, at 6 weekspost-first dose, and at twelve weeks post-first dose. Organ weights weredetermined at study end.

Administration of the AGT dsRNA agent was effective at silencing AGT inboth the high fat and normal chow animals with sustained knockdown ofabout 93% across AGT dsRNA agent treatment groups starting at the firsttime point, two weeks after the first administration of the dsRNA agent.

Treatment with the AGT dsRNA agent was effective at significantlydecreasing weight gain as compared to the PBS treated DIO mice, asdetermined by two-way repeated measures ANOVA in the DIO mice, startingat two weeks post first dose and maintained throughout the study (FIG.8A). In an analysis comparing starting weights, the DIO+AGT dsRNA groupdid not gain weight relative to starting weight until the lasttime-point. Prior to the final time point, mice either lost weightrelative to start weight (weeks 2, 4, 6), or there was no difference inweight (weeks 8, 10). No significant difference in weight was observedbetween the PBS and AGT dsRNA agent chow fed mice until weeks 10 and 12of the study.

Organ weights were determined at the end of the study to assess theeffect of treatment with the AGT dsRNA agent on the location of fatdeposition. Liver weights of the AGT dsRNA agent treated DIO mice weresignificantly lower than the PBS treated DIO mice (FIG. 8B). Nosignificant difference in liver weight was observed between the AGTdsRNA agent and PBS treated normal chow fed mice. Adipose tissue(epidydymal) weight was statistically higher in the DIO+AGT siRNA groupthan the DIO+PBS, while the opposite was true for the normal-weightanimals. There was no significant difference in calf muscle weightsacross all four groups.

Glucose tolerance tests were performed at week 0 (predose), week 6, andweek 12 using a standard protocol. Blood glucose was measured atpredose, 30, 60, 90, and 120 minutes post bolus intraperitoneal glucosedose administration using an AlphaTRAK®2 glucometer (Abbott AnimalHealth). The results are shown in FIGS. 9A-9C. At week 0, DIO mice haddecreased glucose tolerance as compared to the chow fed controls. By sixweeks, a significance difference was observed in multiple comparisonspost-test between the AGT dsRNA agent treated DIO mice and the PBStreated DIO mice. At twelve weeks, and excluding one DIO+PBS animalwhose values were above the glucometer limit, there continued to be asignificant difference between AGT dsRNA agent treated DIO mice and thePBS treated DIO mice. Additionally, the data from DIO+AGT siRNA groupwere not different from either control group (AGT dsRNA agent treated orPBS treated chow fed mice) at six or twelve weeks.

Example 8. Treatment of NASH with an AGT dsRNA Agent in a High Fat HighFructose Mouse Model

A high fat-high fructose (HF HFr) fed mouse model of NASH (Softic et al.J Clin Invest 127 (11):4059-4074, 2017, incorporated herein byreference) was used to demonstrate the efficacy of AGT siRNA to treatNASH and signs of metabolic disorder.

Six to eight weeks-old C57BL/6 male mice obtained from JacksonLaboratories were fed a high fat diet containing 60% of calories as fatplus 30% fructose in water (Hf Hfr diet) for 12 weeks prior to treatmentwith an AGT dsRNA agent or PBS (control) in order to induce NASH or feda standard chow and water diet. Food and water were provided ad libitum.Starting at week 12, HF HFr fed mice were subcutaneously administered a10 mg/kg dose of a dsRNA agent targeted to AGT every other week for atotal of four doses. Two weeks after the final dose (at week 20), liverswere harvested, RNA was isolated, and AGT knockdown in liver wasdetermined by RT-qPCR using the method described above. A 93% decreasein liver AGT mRNA was observed in the AGT dsRNA agent treated Hf Hfr fedmice as compared to the PBS treated HF HFr fed mice.

As expected, body weight (FIG. 10A), cumulative weight gain (FIG. 10B),and terminal liver weight were significantly higher in the HF HFr fedcontrol mice as compared to chow fed mice at all time points. Treatmentwith the AGT dsRNA agent in the HF HFr mice lost weight from week 12till week 20 which resulted in a significant decrease in terminal bodyweight as compared to the control treated mice (p=0.0023). Nosignificant difference in terminal liver weights was observed.

Serum and liver lipids and glucose, and serum insulin were assessed todetermine the effect of the dsRNA agent in the HF HFr model. At week 20,serum triglycerides and glucose levels were substantially the sameacross all three groups (normal chow, HF HFr dsRNA, HF HFr control).Serum cholesterol and insulin levels were about the same in both HF Hfrgroups, and significantly higher than in the chow fed group, asexpected. Treatment with the AGT dsRNA agent was demonstrated tosignificantly decrease serum non-esterified fatty acids (NEFA) (p=0.01).In liver, cholesterol was elevated in both HF HFr groups as compared tonormal chow control, but again, no significant decrease was observedafter treatment with the AGT dsRNA agent. A significant decrease inliver triglycerides (p=0.017) and free fatty acids (p=0.001) wasobserved in the AGT dsRNA agent treated group as compared to the controltreated group. A possible trend towards decreased thiobarbituric acid(TBA), an indicator of lipid oxidation, was seen in the AGT dsRNAtreated group.

Liver injury was indicated by a significant increase in serum alanineaminotransferase (ALT), aspartate aminotransferase (AST), and glutamatedehydrogenase (GLDH) levels in the control treated Hf Hfr mice ascompared to chow fed mice. Treatment with the AGT dsRNA agent resultedin a significant decrease in ALT (p=0.01) (FIG. 11A) with a trendtowards decreased AST (FIG. 11B) and GLDH (FIG. 11C) as compared tocontrol treated HF HFr mice.

Liver injury was also assessed by histopathology and NAS scores. Asexpected, the HF HFr diet induced significant steatosis, balloondegeneration, and lobular inflammation resulting in an increase in theoverall NAS score as compared to chow fed mice. Treatment with the AGTdsRNA agent resulted in a significant decrease in balloon degeneration(p=0.04) with a trend towards decreased lobular inflammation resultingin a significant decrease in the overall NAS score (p=0.01) in the AGTdsRNA agent treated HF HFr fed animals as compared to the controltreated HF HFr fed animals.

These data demonstrate that treatment with the AGT dsRNA agent iseffective in ameliorating some of the signs of NASH. Notably, treatmentwith the AGT dsRNA agent was effective in reducing weight and liverinjury enzymes, with a significant reduction in ALT and a slightreduction in AST and GLDH. Reductions in lobular inflammation andballooning scores were also observed, bringing down the overall NASscore.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

We claim:
 1. A double stranded RNA (dsRNA) agent, or a salt thereof, forinhibiting expression of angiotensinogen (AGT) in a cell, comprising asense strand and an antisense strand forming a double stranded region,wherein the sense strand comprises the nucleotide sequence5′-gsuscaucCfaCfAfAfugagaguaca-3′ (SEQ ID NO:482) and the antisensestrand comprises the nucleotide sequence5′-usGfsuac(Tgn)cucauugUfgGfaugacsgsa-3′ (SEQ ID NO:666), wherein a, g,c, and u are 2′-O-methyl (2′-OMe) A, G, C, and U, respectively; Af, Gf,Cf and Uf are 2′-fluoro A, G, C and U, respectively; s is aphosphorothioate linkage; and (Tgn) is a thymidine-glycol nucleic acid(GNA) S-Isomer.
 2. The dsRNA agent, or salt thereof, of claim 1, furthercomprising a ligand.
 3. The dsRNA agent, or salt thereof, of claim 2,wherein the ligand is conjugated to the 3′ end of the sense strand ofthe dsRNA agent, or salt thereof.
 4. The dsRNA agent, or salt thereof,of claim 2, wherein the ligand is an N-acetylgalactosamine (GalNAc)derivative.
 5. The dsRNA agent, or salt thereof, of claim 2, wherein theligand is one or more GalNAc derivatives attached through a monovalent,bivalent, or trivalent branched linker.
 6. The dsRNA agent, or saltthereof, of claim 4, wherein the ligand is


7. The dsRNA agent, or salt thereof, of claim 6, wherein the dsRNAagent, or salt thereof, is conjugated to the ligand as shown in thefollowing schematic

and, wherein X is O or S.
 8. The dsRNA agent, or salt thereof, of claim7, wherein the X is O.
 9. A double stranded RNA (dsRNA) agent, or a saltthereof, for inhibiting expression of angiotensinogen (AGT) in a cell,comprising a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises the nucleotidesequence 5′-gsuscaucCfaCfAfAfugagaguaca-3′ (SEQ ID NO:482) and theantisense strand comprises the nucleotide sequence5′-usGfsuac(Tgn)cucauugUfgGfaugacsgsa-3′ (SEQ ID NO:666), wherein a, g,c, and u are 2′-O-methyl (2′-OMe) A, G, C, and U, respectively; Af, Gf,Cf and Uf are 2′-fluoro A, G, C and U, respectively; s is aphosphorothioate linkage; and (Tgn) is a thymidine-glycol nucleic acid(GNA)S-Isomer; and wherein the 3′-end of the sense strand is conjugatedto a ligand as shown in the following schematic

wherein X is O.
 10. The dsRNA agent, or a salt thereof, of claim 9,which is in a salt form.
 11. A pharmaceutical composition for inhibitingexpression of a gene encoding AGT comprising the dsRNA agent, or saltthereof, of claim
 1. 12. A pharmaceutical composition for inhibitingexpression of a gene encoding AGT comprising the dsRNA agent, or saltthereof, of claim
 9. 13. A double stranded RNA (dsRNA) agent, or a saltthereof, for inhibiting expression of angiotensinogen (AGT) in a cell,comprising a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand consists of the nucleotidesequence 5′-gsuscaucCfaCfAfAfugagaguaca-3′ (SEQ ID NO:482) and theantisense strand consists of the nucleotide sequence5′-usGfsuac(Tgn)cucauugUfgGfaugacsgsa-3′ (SEQ ID NO:666), wherein a, g,c, and u are 2′-O-methyl (2′-OMe) A, G, C, and U, respectively; Af, Gf,Cf and Uf are 2′-fluoro A, G, C and U, respectively; s is aphosphorothioate linkage; and (Tgn) is a thymidine-glycol nucleic acid(GNA)S-Isomer; and wherein the 3′-end of the sense strand is conjugatedto a ligand as shown in the following schematic

wherein X is O.
 14. The dsRNA agent, or a salt thereof, of claim 13,which is in a salt form.
 15. A pharmaceutical composition for inhibitingexpression of a gene encoding AGT comprising the dsRNA agent, or saltthereof, of claim 13.